NAP1L1 regulates NF-κB signaling pathway acting on anti-apoptotic Mcl-1 gene expression

    NAP1L1 regulates NF-κB signaling pathway acting on anti-apoptotic Mcl-1 gene expression Toshiaki Tanaka, Yasukazu Hozumi, Mitsuyoshi Iino, Kaoru Goto PII: DOI: Reference:

S0167-4889(17)30184-2 doi:10.1016/j.bbamcr.2017.06.021 BBAMCR 18134

To appear in:

BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

16 January 2017 28 June 2017 30 June 2017

Please cite this article as: Toshiaki Tanaka, Yasukazu Hozumi, Mitsuyoshi Iino, Kaoru Goto, NAP1L1 regulates NF-κB signaling pathway acting on anti-apoptotic Mcl-1 gene expression, BBA - Molecular Cell Research (2017), doi:10.1016/j.bbamcr.2017.06.021

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Re-evised manuscript (Manuscript No.: BBAMCR-17-35) Title:

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NAP1L1 regulates NF-B signaling pathway acting on anti-apoptotic Mcl-1 gene expression

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Authors: Toshiaki Tanaka1,*, Yasukazu Hozumi1, Mitsuyoshi Iino2, Kaoru Goto1,*

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Affiliations: 1 Department of Anatomy and Cell Biology, 2 Department of Dentistry, Oral and Maxillofacial Plastic and Reconstructive Surgery, Yamagata University School of Medicine, Yamagata 990-9585, Japan

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* Corresponding author: Toshiaki Tanaka Department of Anatomy & Cell Biology, Yamagata University School of Medicine

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Iida-Nishi 2-2-2, Yamagata 990-9585, JAPAN Tel.: +81-23-23-628-5207 FAX: +81-23-23-628-5210 E-mail: [email protected]

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Kaoru Goto Department of Anatomy & Cell Biology, Yamagata University School of Medicine Iida-Nishi 2-2-2, Yamagata 990-9585, JAPAN Tel.: +81-23-23-628-5207 FAX: +81-23-23-628-5210 E-mail: [email protected]

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Abbreviations:

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NF-κB: Nuclear factor-B; Bcl-2: B cell lymphoma protein 2; Bcl-xl: Bcl-2-related protein long form of Bcl-x; Mcl-1: Myeloid cell leukemia 1; Bax: bcl-2-associated X protein; NAPs: nucleosome assembly proteins; NAP1L: NAP1 like protein;

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DGKdiacylglycerol kinase zeta; siRNA: small interfering RNA; CHX: Cycloheximide; TNF-: Tumor Necrosis Factor-; TMRM: tetramethylrhodamine methyl; PARP: poly ADP-ribose polymerase; IB: inhibitors of NF-B alpha; IKK: IB kinase;ψm: Mitochondrial membrane potential; ChIP: Chromatin Immunoprecipitation; RT-PCR: Reverse Transcription Polymerase Chain Reaction; MOMP: mitochondrial outer membrane permeabilization; CAF-1: Chromatin assembly factor 1; Smarca4: SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4; Kat5: K(Lysine) Acetyltransferase 5; Tip60: HIV-1 Tat interacting protein, 60kDa; Foxa2: forkhead box A 2; Bak: Bcl-2-antagonist killer; Bak: Bcl-2-antagonist killer; BH3: Bcl-2 homology 3

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Abstract

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Nuclear factor-B (NF-κB) participates in apoptosis signaling pathway under various pathophysiological conditions. It exerts transcriptional control on the anti-apoptotic Bcl-2 family, such as Bcl-2, Bcl-xl, and Mcl-1, which act on the mitochondrial outer

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membrane. Previously, we described that NF-B is negatively regulated by diacylglycerol kinase  (DGK), an enzyme that phosphorylates a lipid second messenger diacylglycerol. DGKdownregulation enhances inhibitors of NF-B (IB degradation and p65 subunit phosphorylation, leading to enhanced NF-κB transcriptional activity. Transcriptional machinery is tightly regulated by assembly/disassembly and modification of nucleosomal components. Of those, the human NAP1-like protein (NAP1L) family functions in the transport, assembly/ disassembly of nucleosome core particles. We previously identified NAP1L1 and NAP1L4 as novel DGK binding partners, but the mechanism by which NAP1Ls are

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involved in NF-B signaling pathway remains unclear. Here we show that knockdown of NAP1L1 suppresses IB degradation and nuclear transport of p65 subunit after treatment with TNF-stimulation,leading to attenuation of the NF-B transcriptional activity, whereas NAP1L4 knockdown remains silent. Moreover, ChIP assay reveals that NAP1L1 knockdown attenuates p65 binding to the Mcl-1 promoter after

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TNF-stimulation. This attenuation leads to reduced expression of anti-apoptotic Mcl-1, thereby decreasing the mitochondrial membrane potential and subsequent apoptosis after treatment with TNF-and CHX. Collectively, results of this study suggest that NAP1L1 downregulation renders the cell vulnerable to apoptotic cell death through

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attenuation of NF-B transcriptional activity on the anti-apoptotic Mcl-1 gene. Keywords:

NAP1L1, NAP1L4, NF-B, Mcl-1, TNF-

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1. Introduction

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Transcriptional factor nuclear factor-B (NF-κB) regulates widely various genes that play important roles in signal transduction [1]. When NF-κB is activated by various stimuli, NF-κB induces the expression of genes affecting diverse biological processes including development, immunity, tissue homeostasis, inflammation, stress responses, cell survival, and proliferation [2-6]. In this regard, the specificity and temporal control of gene expression are of crucial physiological interest. Breakdown of NF-κB activity is

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found for several diseases such as cancer, arthritis, chronic inflammation, asthma, neurodegenerative disorders, and heart diseases [7, 8]. Especially, NF-κB controls apoptotic signaling pathways in various tumors [9-11]. It engenders upregulation of B cell lymphoma protein 2 (Bcl-2), Bcl-2-related protein long form of Bcl-x (Bcl-xl), and Myeloid cell leukemia 1 (Mcl-1), along with decreased expression of Bcl-2-associated X protein (Bax) [3, 12, 13], thereby promoting cell survival. Of anti-apoptotic Bcl-2 family members, Mcl-1 was originally isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte and macrophage pathway [14]. Mcl-1 interacts with and

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antagonizes pro-apoptotic Bcl-2 family members on the mitochondrial membrane [15, 16], thereby inhibiting the apoptotic pathway induced by a number of cytotoxic stimuli [17]. Because Mcl-1gene is one of the most highly amplified genes in various human tumors [18], Mcl-1 might be involved in tumor cell development and the mechanism of oncogenesis [19]. Furthermore, the expression of Mcl-1 links to chemotherapeutic resistance and relapse [20, 21]. Therefore, Mcl-1 appears to be a critically important survival factor in many human cancers [19]. Nucleosome assembly proteins (NAPs) influence chromatin compaction and modification. The human NAP1-like protein (NAP1L) family comprises NAP1L1, NAP1L2, NAP1L3, NAP1L4, NAP1L5, and NAP1L6 [22]. Whereas NAP1L1 and L4 are expressed ubiquitously in human tissues, NAP1L2, L3, and L5 are expressed predominantly in the brain [22]. A recent genetic study reveals that NAP1L1 and L4 are highly conserved compared with NAP1L2, L3, and L5 [23], suggesting a fundamental significance of NAP1L1 and L4 in cellular functions. In this regard, we previously demonstrated that NAP1L1 and NAP1L4 associate with diacylglycerol kinase  (DGK, an enzyme that phosphorylates a lipid second messenger diacylglycerol (DG). Both NAP1L1 and NAP1L4 are shown to be involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGK [24], which is implicated in the regulation of p53 function [25]. In addition, our recent study revealed that

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DGKnegatively regulates NF-κB signaling pathway: knockdown of DGK facilitates IB degradation and subsequent nuclear transport of p65, together with p65 phosphorylation, thereby leading to upregulation of NF-B transactivation activity [26]. However, whether NAP1Ls are involved in the regulation of NF-κB transcriptional activity, and how they are involved, remain unclear. This study was conducted to elucidate this point and to examine the regulatory mechanism of NAP1Ls on NF-κB-regulated anti-apoptotic Bcl-2 family members.

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2. Materials and methods

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2.1. Cell culture

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The human cervix carcinoma cell line HeLa cells, the human lung adenocarcinoma epithelial cell line A549 cells, and the human breast cancer cell line MCF7 cells were maintained in Dulbecco’s modified Eagle’s medium (Wako, Osaka, Japan) supplemented with 10% heat-inactivated foetal bovine serum and 100 U/ml penicillin

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and streptomycin. Cell cultures were grown at 37C in a humidified atmosphere of 5% CO2 for routine growth.

2.2. Reagents

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Cycloheximide (CHX) was obtained from Sigma. Tumor Necrosis Factor- (TNF- and tetramethylrhodamine methyl (TMRM) reagent were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany) and Invitrogen (Carlsbad, CA, USA),

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respectively.

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2.3. RNA interference

Two sets of small interfering RNA (siRNA) duplexes for human NAP1L1 (5′-GGCAGACAUUGACAACAAATT-3′, siNAP1L1#1) and NAP1L4 (5′-GAAAGUAUGCAGCGCUAUATT-3′, siNAP1L4#1) were described previously [24]. Another two sets of small interfering RNA (siRNA) duplexes for human NAP1L1 (HSS106946; 5′-GGUAGAAACACCAACAGGAUACAUU-3′, siNAP1L1#2) and NAP1L4 (HSS106956; 5′-CCUUUGAAGGUCCUGAGAUUGUGGA-3′, siNAP1L4#2) were used to knockdown expression of human NAP1L1 and NAP1L4, respectively. Two sets of siRNA duplexes for Mcl-1 were used to knockdown expression of human Mcl-1 (HSS181043 5′-GGUUUGUGGAGUUCUUCCAUGUAGA-3′). Stealth RNAi™ siRNA Negative Control (Invitrogen) was used as a control. Cells were transfected with RNAi duplexes by using Lipofectamine RNAiMAX (Invitrogen). Experiments were performed 48 h after transfection.

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2.4. Immunoblot analysis

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Transfected cells were lysed in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM Na3VO4, 50 mM NaF, 1% Triton X-100, and protease inhibitor cocktail). Protein concentration was determined using BCA Protein Assay Reagent (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The samples were boiled for 10 min

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in SDS sample buffer (New England Biolabs, Inc., Beverly, MA, USA). Equal amounts of protein lysate were separated using SDS-PAGE and electrophoretically transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were then blocked with 5% skim milk in PBS containing Tween 20. The PVDF membranes were immunoblotted using primary antibodies against NAP1L1 [24], NAP1L4 [24],

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DGK [24], p65 (1:1000; 8242; Cell Signalling Technology, Danvers, MA, USA), inhibitors of NF-B alpha (IB (1:1000; 3039; Cell Signalling Technology), phospho-IB (Ser32) (1:1000; 2859; Cell Signalling Technology), acetyl p65 (Lys310) , phospho p65 (Ser536), cleaved poly-ADP-ribose polymers (PARP) (1:1000;

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9541; Cell Signaling), Bcl-xl (1:1000; 2764; Cell Signalling Technology), Bcl-2 (1:1000; 2870; Cell Signalling Technology), Mcl-1 (1:1000; 5453; Cell Signalling Technology), and β-actin (1:5000; 3700; Cell Signalling Technology). Immunoreactive complexes were visualized using the chemiluminescent HRP Substrate ImmobilonTM Western (Millipore).

2.5. Immunofluorescence microscopy Cells were cultured on micro-cover glasses and rinsed with PBS. Cells were fixed with 4% paraformaldehyde, washed with PBS three times, and permeabilized using 0.1% Triton X-100/PBS. Cells were incubated in 10% normal donkey serum for blocking and incubated with p65 antibody overnight at room temperature in a humid chamber. For immunofluorescence analysis, cells were washed with PBS three times and incubated with anti-rabbit IgG-conjugated Alexa 488 for 1 h. TO-PRO-3 was used as a nuclear staining. Images were obtained using a confocal laser-scanning microscope (LSM-700, Carl Zeiss, Jena, Germany).

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2.6. Mitochondrial membrane potential (ψm) measurement assay

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For the ψm measurement assay, we referred and modified a previous protocol [27]. HeLa cells transfected with either of negative control, NAP1L1 or NAP1L4 siRNA were seeded in 96-well plates at optimal density of 1 × 104 cells per well. After cells

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were serum starved and treated with or without TNF-for 4 h, ψm dependent dye TMRM (1 M) was added to each well. After incubation for 60 min, absorbance (A) was measured on a microplate reader (SUNRISE REMOTE, Wako) at 570 nm. Δψm

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measurement was calculated according to the formula: (ATNF- 4h+/ATNF--).

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2.7. Chromatin Immunoprecipitation (ChIP) assay

ChIP assay was performed using the SimpleChIP® Enzymatic Chromatin IP Kit

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(Cell Signalling Technology) according to the manufacturer's instructions. Briefly, HeLa cells were transfected with either of negative control, NAP1L1 or NAP1L4 siRNA. After 48 h, cells (4 × 106 cells) were treated with 1% formaldehyde to cross-link DNA

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binding proteins to genome DNA and cross-linking was stopped by the addition of glycine. After washing with ice-cold PBS several times, the cells were resuspended and added 5 µl of Micrococcal Nuclease to digest DNA to length of 150-900 bp. After stopping digestion by adding 0.5 M EDTA, cells were sonicated to break nuclear membrane by Sonifier 250A (Branson, Danbury, CT, USA). Each 10 µg of chromatin DNA was immunoprecipitated with rabbit IgG or anti-p65 antibody (#8242; Cell Signalling Technology) at 4°C overnight. After incubation, 30 µl of ChIP grade protein G magnetic Beads were added to immunoprecipitating complexes and incubated for 2 h at 4°C. The immunoprecipitating complex was eluted with 150 µL of ChIP elution buffer and formaldehyde cross-linking was reversed by adding NaCl and Proteinase K and by heating at 65°C for 2 h. Genomic DNA was purified and analyzed by PCR. The PCR primer sets were as follows: Bcl-xl (5′-GATCCCCATGGCAGCAGTAAAGCAAG-3′ and 5′-CCCCATCCCGGAAGAGTTCATTCACT-3′), Mcl-1 (5′-CACTTCTCACTTCCGCTTCC-3′ and 5′-TTCTCCGTAGCCAAAAGTCG-3′ ).

2.8. Dual reporter assay

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Cells were transfected with either of negative control, NAP1L1 or NAP1L4 siRNA

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using Lipofectamine RNAimax. After 24 h, cells were transfected with pNF-B-Luc and pRL (renilla luciferase)-null plasmid (internal control) using Lipofectamine 2000. After 24 h plasmid transfection, cells were serum starved and treated with or without

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TNF-for 4 or 6 h. Cell lysates were collected and assayed for luciferase activity using the Dual Luciferase Reporter Assay System (TOYO B-Net, Tokyo, Japan). All luciferase values were normalized to renilla luciferase values (internal control) and expressed as fold induction compared to negative control siRNA. Luminescence was

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measured using a luminescencer-Octa (Atto, Tokyo, Japan).

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2.9. WST-1 assay

Cells were transfected with either of negative control, siNAP1L1 or siNAP1L4

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siRNA and seeded in 96-well plates at optimal density of 4  104 cells per well. Cells were serum starved and treated with or without TNF-and CHX for 4 h. The cell proliferation reagent WST-1 was added to each well, as specified by the supplier (Roche

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Applied Science, Penzberg, Germany). After 4 h of incubation, absorbance (A) was measured on a microplate reader (SUNRISE REMOTE, Wako) at 450 nm. Wells without cells containing complete medium and WST-1 reagent acted as a blank. The

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viability index was calculated according to the following formula: (ATNF- + CHX 4 h+ /A TNF- + CHX 0 h)  100.

2.10. Statistical analysis Data are expressed as means ± standard deviation (SD) from three or more independent experiments. Statistical analysis was performed using Student's t-tests at a significance level of P < 0.01 (**) or P < 0.05 (*) or n.s. (not statistically significant).

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3. Results

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3.1. Knockdown of NAP1L1, but not NAP1L4, inhibits IB degradation

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Our previous study demonstrated that that downregulation of DGK facilitates IBdegradation, thereby enhancing nuclear translocation of NF-B p65 subunit and its transcriptional activity upon TNF- stimulation [26]. Since NAP1L1 and L4 were identified as novel DGK partners [24], we investigated functional roles of these

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molecules in the regulation of NF-B. In the absence of TNF- stimulation, IBexpression did not change in HeLa cells transfected with siRNA for negative control (siControl), NAP1L1 (siNAP1L1) or NAP1L4 (siNAP1L4) (Fig. 1). Results

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showed that after 30 min of TNF- stimulation, NAP1L1 knockdown inhibits IB degradation compared with the control (Fig. 1). Moreover, NAP1L4 knockdown had no

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significant influence on the expression level of IB (Fig. 1). NAP1L1 knockdown inhibited IB degradation whereas NAP1L4 knockdown did not inhibit IB degradation after 10 or 20 min of TNF- stimulation (Fig. S1). We confirmed that similar results were obtained in A549 cells (Fig. S2) and using another two sets of siRNA duplexes for human NAP1L1 and NAP1L4 in HeLa cells (Fig. S3). These NAP1L1

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results indicate that TNF-stimulation.

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3.2. Depletion of NAP1L1 delays the nuclear translocation of p65 Because IBdegradation promotes the nuclear translocation of NF-B heterodimers including p65 subunit into the nucleus in HeLa cells after TNF- treatment [28, 29], we examined whether NAP1L1 knockdown may influence the dynamics of NF-B p65 subcellular localization in response to TNF-. NF-B p65 subunit was localized primarily to the cytoplasm of cells transfected with either siControl, siNAP1L1, or siNAP1L4 under normal conditions without TNF- (Fig. 2A). After 30 min of stimulation, p65 subunit was found to be accumulated mostly in the nucleus of cells transfected with siControl and siNAP1L4, although it remained largely cytoplasmic in cells transfected with siNAP1L1 (Figs. 2A and 2B). Furthermore, we confirmed these morphological data by subcellular fractionation and immunoblot analysis (Fig. 2C). In this regard, we examined whether NAP1L1 and NAP1L4 associate with NF-B p65 subunit. Immunoprecipitation assay revealed that NAP1Ls do not associate with p65

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subunit in the presence or absence of TNF- stimulation (data not shown). Taken together, these data indicate that NAP1L1 knockdown delays nuclear translocation of

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p65 subunit by suppressing IBdegradation after TNF- treatment.

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3.3. NAP1L1 knockdown suppresses IB phosphorylationat Ser 32, p65 acetylation at Lys310, and p65 phosphorylation at Ser536 Reportedly, IκBα phosphorylation at Ser32 is necessary for its own degradation [30].

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Because we found that NAP1L1 knockdown suppresses IB degradation after TNF- treatment (Fig. 1), we investigated whether NAP1L1 knockdown alters this

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phosphorylation level after TNF- stimulation. As depicted in Fig. 3, NAP1L1 knockdown decreased the Ser32 phosphorylation of IκBα compared with the control

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after stimulation (Fig. 3), which suggests that NAP1L1 knockdown attenuates IB phosphorylation at Ser32, thereby leading to suppressed IB degradation and cytoplasmic retention of the p65 subunit. Similar results were obtained in A549 cells (Fig. S2) and using another two sets of siRNA duplexes for human NAP1L1 and

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NAP1L4 in HeLa cells (Fig. S3). In this regard, p65 subunit is shown to be acetylated in the nucleus after translocation [31]. Therefore, we next asked whether p65 acetylation is altered in

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siNAP1L1-treated cells Consistent with the data that p65 subunit translocates to the nucleus in cells transfected with siControl or siNAL1L4 after 30 min of TNF- stimulation (Figs. 2A and 2B), p65 acetylation at Lys310 was also increased significantly (Fig. 3). This acetylation was less pronounced in cells transfected with siNAP1L1 (Fig. 3), which coincides with attenuated nuclear translocation of p65 (Figs. 2A and 2B). Moreover, similar results were obtained in A549 cells (Fig. S2) and using another two sets of siRNA duplexes for human NAP1L1 and NAP1L4 in HeLa cells (Fig. S3). Reportedly, NF-B transcriptional activity is upregulated by phosphorylation of p65 at Ser536 [32, 33]. Although the phosphorylation levels of p65 at Ser536 remained unchanged upon transfection with either siControl, siNAP1L1 or siNAP1L4 at 30 min after TNF- stimulation (data not shown), we found that the p65 phosphorylation of this site was downregulated in cells transfected with siNAL1L1 at the time point of 10 min after TNF- stimulation (Fig. S4).

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3.4. Depletion of NAP1L1 suppresses NF-B transcriptional activity after TNF- stimulation 

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Having shown that NAP1L1 knockdown delays p65 nuclear translocation (Fig. 2) and decreases its acetylation at Lys310 (Fig. 3), we next used luciferase assay to investigate

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whether downregulation of NAP1L1 affects NF-B transcriptional activity (Fig. 4). Compared to cells transfected with siControl, NF-B transcriptional activity was decreased to less than half in cells transfected with siNAP1L1 after 4 h (Fig. 4) or 6 h

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(Fig. S5) of TNF- stimulation. However, NF-B transcriptional activity showed no apparent change in cells transfected with siNAP1L4 (Fig. 4 and Fig. S5). Similar results were obtained using another siNAP1L1 and siNAP1L4 oligos (Fig. S6A) and in A549

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cells (Fig. S6B). These results show that NAP1L1 knockdown suppresses NF-B transcriptional activity, although NAP1L4 knockdown exhibits no significant effect on its activity.

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3.5. NAP1L1 knockdown attenuates the expression of Mcl-1 after TNF- stimulation

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NF-κB activation induces the expression of several anti-apoptotic genes [9-11], including Bcl-2, Mcl-1, and Bcl-xl [3, 12, 13, 34]. Therefore, we performed immunoblot analysis to investigate whether NAP1L1 knockdown affects the expression of these anti-apoptotic genes. Intriguingly, of those genes, Mcl-1 expression was

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selectively attenuated in cells transfected with siNAP1L1 after 4 h TNF- stimulation (Fig. 5, lane 5 vs. lanes 4 and 6). However, no significant difference was found in the expression of those genes in cells transfected with siNAP1L4 (Fig. 5, lane 5 vs. lanes 4 and 6).

3.6. NAP1L1 knockdown attenuates p65 binding to the Mcl-1 promoter after TNF- stimulation Because NAP1L1 knockdown suppressed NF-B transcriptional activity (Fig. 4 and Fig. S6) and specifically attenuated Mcl-1 expression after TNF- stimulation (Fig. 5, lane 5 vs. lanes 4 and 6), we next examined whether NAP1L1 knockdown affects the binding of p65 to Mcl-1 promoter. To this end, we performed chromatin immunoprecipitation (ChIP) assay using anti-p65 antibody in siNAP1L1-transfected HeLa cells. Results

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showed that p65 binding to the Mcl-1 promoter is attenuated under conditions of

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NAP1L1 knockdown after TNF- stimulation (Fig. 6, lower panel, lane 6 vs. lane 12) whereas control knockdown rather increases its binding (Fig. 6, lower panel, lane 3 vs. lane 9). On the other hand, p65 binding to the Bcl-xl promoter increased under

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conditions of both control and NAP1L1 knockdown after TNF- stimulation (Fig. 6, upper panel, lane 3 vs. lane 9, lane 6 vs. lane 12). These results confirmed that NAP1L1 positively modulates Mcl-1 expression at the transcriptional level.

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3.7. NAP1L1 knockdown disrupts mitochondrial membrane potential, thereby promoting apoptosis The anti-apoptotic Bcl-2 proteins antagonize mitochondrial outer membrane permeabilization (MOMP) and maintain mitochondrial membrane potential (Δψm) [35]. Because Mcl-1 expression was attenuated in cells transfected with siNAP1L1 after

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TNF- treatment (Fig. 5), we sought to ascertain whether Δψm is changed in HeLa cells transfected with siNAP1L1, or not. As portrayed in Fig. 7A, NAP1L1 knockdown

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disrupted Δψm after TNF- stimulation compared with the control and NAP1L4 knockdown. We also confirmed this result using A549 cells (Fig. S7). As a next step, we assessed the effect of NAP1L1 downregulation on apoptosis. Apoptosis is induced after TNF- stimulation combined with CHX [36]. We first confirmed that Mcl-1 knockdown decreases cell viability after treatment with

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TNF-and CHX (Fig. S8). As portrayed in Fig. 7B, the level of cleaved PARP, an apoptosis marker, was increased after 4 h stimulation in cells transfected with siNAP1L1 compared with siControl or siNAP1L4. Similar results were obtained for MCF7 and A549 cells (Fig. S9A and S9B). We also conducted immunofluorescence analysis using antibody against caspase3, a critical apoptosis executioner that is cleaved in cells undergoing apoptosis [37]. As shown in Fig. 7C, the cleaved caspase3 was not detected in cells transfected with either siControl, siNAP1L1, or siNAP1L4 without stimulation (Fig. 7C). We confirmed that the cleaved caspase3 level is increased greatly in cells transfected with siNAP1L1 after stimulation, compared with either siControl or siNAP1L4 (Figs. 7D and 7E). Cells undergoing apoptosis are morphologically characterized by nuclear shrinkage [38], which represents chromatin condensation in an early phase of dying cells. NAP1L1 knockdown increased the number of cells showing nuclear shrinkage by

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more than two-fold after stimulation (Fig. 7D). Furthermore, WST-1 assay confirmed

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quantitatively that cell viability after treatment with TNF-and CHX was decreased by more than 20% under NAP1L1 knockdown conditions (Fig. 7F). Similar results were obtained for A549 cells (Fig. S10) and using another siNAP1L1 oligos (Fig. S11A and S11B). Collectively, the results indicated that NAP1L1 knockdown promotes apoptosis through disruption of Δψm by suppressing Mcl-1 expression.

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3.8. Mcl-1 knockdown does not change the expression of NAP1L1 and NAP1L4 NAP1L1 knockdown attenuated Mcl-1 induction and promoted apoptosis after

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stimulation with TNF-and CHX (Fig. 7). To confirm that Mcl-1 acts downstream of NAP1L1, but not upstream, we examined whether Mcl-1 knockdown influences the expression of NAP1L1 and NAP1L4. As presented in Fig. 8, the expression levels of NAP1L1 and NAP1L4 were not changed by Mcl-1 knockdown (Fig. 8), confirming that NAP1L1 is an upstream regulator of Mcl-1.

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4. Discussion

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NAP1L1 and NAP1L4 belong to the nucleosome assembly protein family [22]. These

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molecules are recently identified as binding partners of DGK [24]. Our earlier study demonstrated that DGK downregulation facilitates NF-B signaling pathway, suggesting involvement of DGK in this pathway [26]. Therefore, we surmised that NAP1L1 and NAP1L4 also play a role in the control of NF-B signaling as participants of the regulatory complex of this transcription factor.

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This study revealed that NAP1L1 knockdown suppresses IB degradation and nuclear translocation of p65 subunit after TNF- stimulation, thereby attenuating the NF-B transcriptional activity Moreover, NAP1L1 knockdown specifically attenuates the expression of Mcl-1 among the NF-B-regulated anti-apoptotic Bcl-2 family after pro-apoptotic stimulation. Because Mcl-1 plays a critically important role in maintaining mitochondrial membrane potential, its downregulation results in mitochondrial dysfunction and subsequent apoptosis, which suggests that NAP1L1 is

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involved in stress response and apoptosis through control of the NF-B signaling pathway acting on the anti-apoptotic Mcl-1 gene.

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Actually, NAP1L1 and NAP1L4 have similar domain structures and functional properties. These molecules serve as histone transporters and play a pivotal role in assembly or disassembly of nucleosome core particles [39-41]. Furthermore, both NAP1L1 and NAP1L4 interact with chromatin assembly factor 1 (CAF-1), suggesting the involvement of histone H3 and H4 in synthesis for DNA replication [42]. How NAP1L1 and NAP1L4 are differentiated in terms of cellular functions remains elusive. An earlier report of the relevant literature suggests that human NAP1L1 shows significantly greater histone disassembly activity than human NAP1L4 [39]. Do they perform the same function with different efficiency and play redundant roles? In this respect, the present study reveals a specific role assigned to NAP1L1 in the regulation of NF-B signaling pathway. NAP1L1 knockdown attenuates the expression of Mcl-1, but not Bcl-2 and Bcl-xl, after TNF-treatment whereas NAP1L4 knockdown remains silent (Fig. 5), suggesting a link between NAP1L1 and Mcl-1-mediated mitochondrial function. The precise mechanism of how only Mcl-1 expression is influenced by NAP1L1 remains elusive. It is generally considered that a given gene is under control of more than one transcription factor with variable efficiency. In the case of NAP1Ls, they promote transcription factor binding to hidden nucleosomal site by elimination of H2A/H2B dimers [43]. Thus, transcription factor binding to nucleosomal site is mediated through elimination of H2A/H2B dimers by

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NAP1Ls. We assume that NAP1L1, but not NAP1L4, removes H2A/H2B dimers to access Mcl-1-specific nucleosomal site. Moreover, nuclear translocated p65/p50 heterodimer is shown to interact with chromatin-modifiers such as histone deacetylases (HDACs), p300 or cAMP response element-binding protein (CREB)-binding protein (CBP) [44-47]. In this regard, NAP1L1 also interacts with p300, thereby forming NAP1L1-p300 complex [48] and stabilizing the binding of sequence-specific transcriptional factors [43]. It is hypothesized that NAP1L1 acts at the Mcl-1-specific nucleosomal site via p300. Further investigation is necessary to clarify this hypothesis.

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Hoffmann et al. reported that chromatin remodeling and NF-B activation in the nucleus is coordinated in response to stimulation and that NF-B induces genes depending on characteristics of stimulation and its severity and duration [49]. At the

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moment, it remains unclear how the nucleosomes affect the interaction of NF-B and DNA binding sites. Natoli suggests that although nucleosomes are transparent to NF-B,

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nucleosomal chains might create efficient barriers to the binding of NF-B and DNA and that the nucleosomes might control NF-B recruitment to block the interaction of NF-B and DNA [50]. In this regard, results of the present study suggest that NAP1L1 is involved in selectivity of NF-B-induced genes through the regulation of the

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accessibility of NF-B and DNA in response to TNF- stimulation. However, the precise mechanism of how NAP1L1 is coordinated to control the interaction of NF-B and DNA binding sites through nucleosomal assembly/disassembly and occupation must be answered in future studies. Several studies reveal that NAP1L1 is highly expressed in some tumors [51-58] and suggest that NAP1L1 might be involved in oncogenesis. In addition, Mcl-1 is highly expressed in various human tumors [18] and regulates tumor development and oncogenesis [19]. These reports suggest strongly that NAP1L1-Mcl-1 axis is a target for tumor suppression. Actually, Bcl-2 family inhibitors such as obatoclax bind to Mcl-1, thereby abolishing an interaction of Mcl-1 with the pro-apoptotic Bcl-2-antagonist killer (Bak) [59]. Recently, ligands and peptides that mimic Bcl-2 homology 3 (BH3) domain and which bind specifically to BH3-binding groove of Mcl-1 are expected to be potential therapeutic agents for tumors [60, 61]. However, the usage of only obatoclax or BH3 mimics have proved to be far from fully effective for certain types of cancer cells [19]. However, deletion of Mcl-1 is reported to induce cell death in tumors [62, 63] because Mcl-1 is necessary for both apoptotic apoptotic resistance and metabolic function for high proliferation of cancer cells [19]. These results of studies suggest that attenuation of Mcl-1 expression is a more promising strategy for cancer cells. In summary, the molecular complex containing DGK and NAP1L1 modulates the

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NF-B signaling pathway bidirectionally, depending on the expression levels of each component. Downregulation of DGK augments the NF-B signaling pathway [26] whereas attenuation of NAP1L1 suppresses this pathway specifically acting on Mcl-1 gene expression. It is possible that inhibitors and compounds targeting NAP1L1 are a more effective therapy for various tumors via the suppression of Mcl-1 expression. Further studies depicting the mechanism for the regulation of NAP1L1-Mcl-1 axis are expected to contribute to the development of new tumor therapies.

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Acknowledgements

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This work was supported by Grants-in-Aid from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (KG).

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Figure legends

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Fig. 1. NAP1L1 knockdown but not NAP1L4 inhibits IB degradation after TNF-treatment. HeLa cells were transfected with control or siNAP1L1#1 or siNAP1L4#1 siRNA. After

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48 h, cells were serum starved and stimulated with TNF-(20 ng/ml) at 30 min. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. Size markers are on the left.

Fig. 2. NAP1L1 knockdown delays nuclear translocation of p65 at 30 min after

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TNF-treatment. (A) HeLa cells were transfected with control, NAP1L1, or NAP1L4 siRNAs. After 48 h,

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cells were serum starved for 4 h and incubated without TNF- or with TNF-(20 ng/mL) for 30 min. Cells were fixed with 4% paraformaldehyde and stained with anti-p65 antibody (green). TO-PRO-3 was used for nuclear staining (blue). Scale bars = 20 µm. (B) The number of p65-positive cells in the nucleus was counted (n = 100) in

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three arbitrary fields. The graph shows the average number of p65-positive cells in the nucleus. Asterisks indicate significance compared with control siRNA, **P < 0.01 (Student’s t-test). (C) HeLa cells were transfected with control or NAP1L1 or NAP1L4

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siRNAs. After 48 h, cells were serum starved for 4 h and incubated with TNF-(20 ng/mL) for 30 min or without TNF-and cells were divided into nuclear and cytoplasmic fractions. Each fraction was analyzed by immunoblotting using the indicated antibodies. Rho GDIα was used as a cytoplasm marker, and Lamin B was used as a nucleus marker. N, nuclear fraction and C, cytoplasmic fraction.

Fig. 3. The depletion of NAP1L1 suppresses IB phosphorylationat Ser 32 and p65 acetylation at Lys310 after TNF-treatment. HeLa cells were transfected with control or NAP1L1 or NAP1L4 siRNA. After 48 h, cells were serum starved and stimulated with TNF-20 (ng/ml) for 30 min. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. β-actin served as the loading control.

Fig. 4. NAP1L1 knockdown suppresses the NF-B transcriptional activity after

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TNF-treatment. HeLa cells were transfected with control or NAP1L1#1 or NAP1L4#1 siRNAs. After 24

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h, cells were co-transfected with either pNF-B or pRL-null (internal control) reporter constructs. After 24 h, cells were serum starved and incubated with TNF- (20 ng/ml) for 4 h or without TNF-. Cell lysates were analyzed for NF-B transcriptional activity using the Dual Luciferase Reporter Assay System. NF-B transcriptional activities were measured in three independent experiments. Histogram data were determined using the

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formula ATNF-+/ATNF-- and are shown as the mean ± SD. Asterisk indicates significance, ** P < 0.01 (Student’s t-test).

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Fig. 5. The depletion of NAP1L1 attenuates the expression of Mcl-1 after TNF- stimulation. HeLa cells were transfected with control or NAP1L1#1 or NAP1L4#1 siRNAs. After 48

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h, cells were serum starved and stimulated with TNF- (20 ng/ml) for 4 or 6 h or without TNF-. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. β-actin served as the loading control.

Fig. 6. NAP1L1 knockdown attenuates p65 binding to the Mcl-1 promoter after TNF- stimulation. HeLa cells were transfected with control or NAP1L1#1 or NAP1L4#1 siRNAs. After 48

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h, cells were serum starved and stimulated with or without TNF-(20 ng/ml) for 4 h. Genomic DNA extracted from HeLa cells was precipitated with anti-p65 antibody or normal rabbit IgG. Precipitated p65 bound DNA was amplified by PCR using the specific primers Bcl-xl (upper panel) and Mcl-1 (lower panel).

Fig. 7. NAP1L1 knockdown disrupts mitochondrial membrane potential, thereby promoting apoptosis. (A) HeLa cells were transfected with control or NAP1L1#1 or NAP1L4#1 siRNAs. After 48 h, cells were serum starved and stimulated with TNF-(20 ng/ml) for 4 h or without TNF-. After cells were serum starved and treated with TNF-for 4 h or without TNF-, 1 M of TMRM was added to each well for 60 min. Cells were analyzed for Δψm measurements using microplate reader at 570 nm. Δψm measurements were measured in three independent experiments. Histogram data were determined

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using the formula ATNF-4h+/ATNF-- and are shown as the mean ± SD. Asterisk indicates significance, **P < 0.01 (Student’s t-test). (B) HeLa cells were transfected with control or NAP1L1#1 or NAP1L4#1 siRNAs. After 48 h, cells were serum starved and

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stimulated with TNF- (20 ng/ml) and CHX (10 g/ml) for 4 h or without. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. -actin served as the loading control. (C, D) HeLa cells were transfected with control or NAP1L1#1 or

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NAP1L4#1 siRNAs. After 48 h, cells were serum starved and stimulated without TNF- and CHX (C) or with TNF- (20 ng/ml) and CHX (10 g/ml) for 4 h (D). Cells were fixed with 4% paraformaldehyde and stained with anti-cleaved caspase3 antibody (green). TO-PRO-3 was used for nuclear staining (blue). Scale bar = 20 µm. (E) The number of cleaved caspase3-positive cells was counted (n = 100) in five arbitrary fields. The average numbers of cleaved caspase3 positive cells are shown. Asterisks indicate significance compared with negative control siRNA. **P < 0.01 (Student’s t-test). n.s., not significant. (F) HeLa cells were transfected with control, NAP1L1#1 siRNA, or NAP1L4#1 siRNAs. After 24 h, cells were seeded in 96-well plates for WST-1 assays

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and treated without or with TNF- (20 ng/ml) and CHX (10 g/ml) for 4 h. Data are shown as the means ± SD of 20 individual samples. Asterisks indicate statistical

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significance. **P < 0.01 (Student’s t-test).

Fig. 8. Mcl-1 knockdown does not change the expression of NAP1L1 and NAP1L4. A549 cells were transfected with control or Mcl-1 siRNAs. After 48 h, cells were serum

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starved and stimulated with TNF- (20 ng/ml) and CHX (10 g/ml) for 4 h or without TNF- and CHX. Whole cell lysates were analyzed by immunoblotting using the indicated antibodies. -actin served as the loading control.

Fig. 9. A schematic model underlying mechanism of Mcl-1 expression through NF-B pathway in the depletion of NAP1L1. A hypothetical model underlying the NF-B regulatory complex containing NAP1L1 and DGK based on the present study and previous report [26]. Knockdown effects of NAP1L1 (upper) and DGK (lower), together with the wild-type control (middle), on the regulation of NF-B pathway are shown. The regulatory complex in the absence of NAP1L1 (upper) inhibits IB degradation, thereby suppressing NF-B p65 nuclear translocation and subsequent transcriptional activity. On the other hand, the regulatory complex devoid of DGK (lower) accelerates IBdegradation, thereby facilitating

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nuclear translocation of p65 subunit and resultant transcription activity.

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There are no conflicts of interest to declare.

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Highlights

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・Depletion of NAP1L1 delays the nuclear translocation of p65. ・ Depletion of NAP1L1 suppresses NF-B transcriptional activity after TNF- stimulation.

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・NAP1L1 knockdown attenuates p65 binding to the Mcl-1 promoter after TNF- stimulation.

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・NAP1L1 knockdown disrupts mitochondrial membrane potential, thereby promoting apoptosis.

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