Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

doi: 10.1096/fj.201902039R

Figure Lengend Snippet: FIGURE 1 HDAC4 and p-HDAC4 are specifically modulated in AT cells by dex. A and B, Typical images illustrating the nuclear localization of HDAC4 and p-HDAC4 in control and dex-treated WT hT and AT 648 hT cells stained by IF. C and D, Quantification of the signals derived from the IF experiments. At least 200 nuclei were counted for data processing. HDAC4 only accumulated in AT 648 hT cells after dex treatment (P = .012 t-test), while no statistical differences were appreciable for p-HDAC4 quantitation. E-I, Western blot analysis on nuclear protein extracts and total protein extracts of all the tested cell lines. The quantitation of the immunoreactive bands is also reported. Nuclear HDAC4 only accumulated in AT 648 hT-treated cells (Wilcoxon test P = .0313 n = 9), while no differences were recorded for nuclear p-HDAC4. The analysis of total protein extracts showed an increment of HDAC4 in AT 648 hT-treated cells (Wilcoxon test P = .313 n = 9), while p-HDAC4 was downregulated in WT hT and upregulated in AT 648 hT-treated cells (Wilcoxon test P = .0216, n = 9)

Article Snippet: The following antibodies were used in the analyses : anti-HDAC4 (Cell Signaling Technology CST, Thermo Fisher Scientific, TFS), anti-phospho HDAC4 Ser632 (CST), anti-NFE2L2 (Santa Cruz Biotechnology, SCBT), anti-HIF1-a (CST, TFS), anti-DDIT4 (Bethyl and CST), anti-4E-BP1 (CST) anti-phospho 4E-BP1 Thr37/46 (CST), anti-p70S6K (CST and Bethyl), anti-phospho p70S6K Thr389 (CST), anti-LC3B (CST), anti-SQSTM1/ p62 (CST), anti-VPS18 (TFS), anti-AKT (CST) anti-phospho AKT Ser473 (CST), anti-GSK-3a/b (CST), and anti-phospho GSK-3a/b Ser21/9 (CST).

Techniques: Control, Staining, Derivative Assay, Quantitation Assay, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

doi: 10.1096/fj.201902039R

Figure Lengend Snippet: FIGURE 2 HDAC4 cysteins are reduced after dex treatment. A, Representative western blot image of the reduced HDAC4 immunoreactive bands obtained by biotin-modified cysteines captured by monomeric avidin beads and probed with anti-HDAC4 antibodies. B, Western Blot quantification. Dex improved the reduced status of HDAC4 only in AT 648 hT cells, promoting its nuclear translocation (P = .0313 Wilcoxon test, n = 5)

Article Snippet: The following antibodies were used in the analyses : anti-HDAC4 (Cell Signaling Technology CST, Thermo Fisher Scientific, TFS), anti-phospho HDAC4 Ser632 (CST), anti-NFE2L2 (Santa Cruz Biotechnology, SCBT), anti-HIF1-a (CST, TFS), anti-DDIT4 (Bethyl and CST), anti-4E-BP1 (CST) anti-phospho 4E-BP1 Thr37/46 (CST), anti-p70S6K (CST and Bethyl), anti-phospho p70S6K Thr389 (CST), anti-LC3B (CST), anti-SQSTM1/ p62 (CST), anti-VPS18 (TFS), anti-AKT (CST) anti-phospho AKT Ser473 (CST), anti-GSK-3a/b (CST), and anti-phospho GSK-3a/b Ser21/9 (CST).

Techniques: Western Blot, Modification, Avidin-Biotin Assay, Translocation Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

doi: 10.1096/fj.201902039R

Figure Lengend Snippet: FIGURE 3 TXN is upregulated by dex in NFE2L2 in an independent manner. A, HDAC4 reduction should be mediated by TXN, which is actually overexpressed upon dex treatment in both WT hT and AT 648 hT cells (Wilcoxon test P = .0239 and P = .041, respectively, n = 5). B, The overexpression of TXN was not mediated by NFE2L2 since no further accumulation in the nucleus is observed after dex treatment in all the analyzed cell lines. However, a higher basal amount of nuclear NFE2L2 was observed in AT cells than in WT cells (Test U Mann-Whitney P = .317, n = 5)

Article Snippet: The following antibodies were used in the analyses : anti-HDAC4 (Cell Signaling Technology CST, Thermo Fisher Scientific, TFS), anti-phospho HDAC4 Ser632 (CST), anti-NFE2L2 (Santa Cruz Biotechnology, SCBT), anti-HIF1-a (CST, TFS), anti-DDIT4 (Bethyl and CST), anti-4E-BP1 (CST) anti-phospho 4E-BP1 Thr37/46 (CST), anti-p70S6K (CST and Bethyl), anti-phospho p70S6K Thr389 (CST), anti-LC3B (CST), anti-SQSTM1/ p62 (CST), anti-VPS18 (TFS), anti-AKT (CST) anti-phospho AKT Ser473 (CST), anti-GSK-3a/b (CST), and anti-phospho GSK-3a/b Ser21/9 (CST).

Techniques: Over Expression, MANN-WHITNEY

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

doi: 10.1096/fj.201902039R

Figure Lengend Snippet: FIGURE 4 HDAC4 interaction with HIF-1a is boosted after dex treatment. A, Representative western blot of HIF-1a and 14.3.3 ζ/δ probed membranes containing HDAC4 co-immunoprecipitated proteins. Dex enhanced HDAC4-HIF-1a interaction in AT 648 hT cells. Weak signals were observed in other lanes. Dex seemed to reduce the HDAC4-14.3.3 ζ interaction in AT 648 hT cells. B, Representative western blot analysis of the HIF-1a nuclear amount from all cell line conditions. Quantitation is also reported and HIF-1a only accumulated in AT 648 hT cells after dex stimulation (Wilcoxon test P < .0313 n = 7). C. HIF-1a nuclear localization (green) was also observed by indirect immunofluorescence imaging of AT cells treated with dex or untreated, and in some loci it colocalizes with HDAC4 (red)

Article Snippet: The following antibodies were used in the analyses : anti-HDAC4 (Cell Signaling Technology CST, Thermo Fisher Scientific, TFS), anti-phospho HDAC4 Ser632 (CST), anti-NFE2L2 (Santa Cruz Biotechnology, SCBT), anti-HIF1-a (CST, TFS), anti-DDIT4 (Bethyl and CST), anti-4E-BP1 (CST) anti-phospho 4E-BP1 Thr37/46 (CST), anti-p70S6K (CST and Bethyl), anti-phospho p70S6K Thr389 (CST), anti-LC3B (CST), anti-SQSTM1/ p62 (CST), anti-VPS18 (TFS), anti-AKT (CST) anti-phospho AKT Ser473 (CST), anti-GSK-3a/b (CST), and anti-phospho GSK-3a/b Ser21/9 (CST).

Techniques: Western Blot, Immunoprecipitation, Quantitation Assay, Immunofluorescence, Imaging

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

doi: 10.1096/fj.201902039R

Figure Lengend Snippet: FIGURE 6 DDIT4 is selectively transcribed in AT cells by the HIF-1a-HDAC4 complex after dex stimulation. A, qPCR quantification of ChIP outcome in AT 468 hT cells revealed that the amount of HIF-1a in the DDIT4 promoter was unaltered (Wilcoxon test P = .14, n = 5), while the amount of HDAC4 was markedly increased after dex treatment (Wilcoxon test P = .0355, n = 5). B-C, The silencing of HIF-1a and HDAC4 by siRNAs, decreased DDIT4 expression in treated AT 648 hT cells by approximately 70% (siRNA HIF-1a, Wilcoxon test P = .0313 n = 7) and by approximately 50% (siRNA HDAC4, Wilcoxon test P = .0084 n = 6) when compared to the siRNA SCR dex-treated control. No differences between siRNA SCR and siRNA HIF-1a or siRNA HDAC4-untreated AT cells were observed. DDIT4 transcription is improved by HDAC4 HIF- 1a stabilization upon dex stimulation specifically in AT cells. D. DDIT4 protein amounts in HIF-1a and HDAC4 targeting siRNAs in all the tested cell lines. DDIT4 protein levels were also reduced in treated AT 648 hT after HIF-1a and HDAC4 silencing. DDIT4 HIF-1a-silenced western blot quantification is reported in Supplemental Figure S6

Article Snippet: The following antibodies were used in the analyses : anti-HDAC4 (Cell Signaling Technology CST, Thermo Fisher Scientific, TFS), anti-phospho HDAC4 Ser632 (CST), anti-NFE2L2 (Santa Cruz Biotechnology, SCBT), anti-HIF1-a (CST, TFS), anti-DDIT4 (Bethyl and CST), anti-4E-BP1 (CST) anti-phospho 4E-BP1 Thr37/46 (CST), anti-p70S6K (CST and Bethyl), anti-phospho p70S6K Thr389 (CST), anti-LC3B (CST), anti-SQSTM1/ p62 (CST), anti-VPS18 (TFS), anti-AKT (CST) anti-phospho AKT Ser473 (CST), anti-GSK-3a/b (CST), and anti-phospho GSK-3a/b Ser21/9 (CST).

Techniques: Expressing, Control, Western Blot

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: DDIT4 gene expression is switched on by a new HDAC4 function in ataxia telangiectasia.

doi: 10.1096/fj.201902039R

Figure Lengend Snippet: FIGURE 10 HDAC4 and DDIT4 are also modulated by dex in AT patients. A, HDAC4 qPCR on patients’ samples, previously tested by microarray analyses, revealed that HDAC4 is downregulated in AT patients compared to healthy subjects, and the gene expression in AT patients who received EryDex was restored (Kruskal-Wallis P = .036 followed by Dunn test). B, Like HDAC4, DDIT4 was downregulated in AT patients compared to healthy subjects (Kruskal-Wallis P = .016 followed by Dunn test), and dex improved DDIT4 mRNA levels only in some treated patients

Article Snippet: The following antibodies were used in the analyses : anti-HDAC4 (Cell Signaling Technology CST, Thermo Fisher Scientific, TFS), anti-phospho HDAC4 Ser632 (CST), anti-NFE2L2 (Santa Cruz Biotechnology, SCBT), anti-HIF1-a (CST, TFS), anti-DDIT4 (Bethyl and CST), anti-4E-BP1 (CST) anti-phospho 4E-BP1 Thr37/46 (CST), anti-p70S6K (CST and Bethyl), anti-phospho p70S6K Thr389 (CST), anti-LC3B (CST), anti-SQSTM1/ p62 (CST), anti-VPS18 (TFS), anti-AKT (CST) anti-phospho AKT Ser473 (CST), anti-GSK-3a/b (CST), and anti-phospho GSK-3a/b Ser21/9 (CST).

Techniques: Microarray, Gene Expression

Journal: Frontiers in Pharmacology

Article Title: Class IIa histone deacetylase inhibition ameliorates acute kidney injury by suppressing renal tubular cell apoptosis and enhancing autophagy and proliferation

doi: 10.3389/fphar.2022.946192

Figure Lengend Snippet: Expression of HDAC4, HDAC5, HDAC7, and HDAC9 in the kidney following ischemia/reperfusion (I/R) and folic acid (FA) injury (A) At 48 h after sham operation or I/R, kidneys were collected and tissue lysates were subjected to immunoblot analysis with antibodies against HDAC4, 5, 7, 9, and GAPDH. Representative immunoblots are three samples in each group (B) Expression levels of HDAC4, 5, 7, 9 were quantified by densitometry and normalized with GAPDH. Data are expressed as means ± SD ( n = 3). ** p < 0.01 (C) At 48 h after sham operation, FA, or I/R, kidneys were collected and tissue sections were subjected to immunohistochemical staining followed by photomicrograph illustrating protein expression of individual class IIa HDACs. Scale bar, 20 µm. Original magnification, ×200.

Article Snippet: Antibodies to HDAC4, HDAC5, HDAC7, and HDAC9 were purchased from Santa Cruz Biotechnology (Dallas, TX, United States).

Techniques: Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: PATJ inhibits histone deacetylase 7 to control tight junction formation and cell polarity.

doi: 10.1007/s00018-023-04994-3

Figure Lengend Snippet: Fig. 2 PATJ localizes to pri- mary cilia and regulates cilia maintenance. A–C Immu- nostainings of PATJ, acetylated α-Tubulin (Ac-Tubulin) and Crb3b in wild-type MDCK cells (A), PATJ-deficient cells (B) and MDCK∆PATJ-cells with mPATJ-GFP rescue (C). Arrows indicate primary cilia. D–F Staining of primary cilia with Arl13b and ac-Tubulin demonstrates a reduction in primary cilia. G Quantification of primary cilia in the indi- cated cell lines. Error bars are standard errors of the means. Significance was determined by one-way ANOVA test and Bon- ferroni correction: *p < 0.05, ns not significant. Scale bars are 10 µm in A–C and 20 µm in D–F

Article Snippet: The following primary antibodies were used: rabbit anti Arl13b (1:200, Proteintech #17711- 1-AP), goat anti-Claudin7 (1:50, Santa Cruz #17670), rat anti-Crb3b (1:100, raised in this study), rat anti-E-Cadherin (1:100, Santa Cruz #59778), goat anti-GFP (1:500, Rockland #600101215), mouse anti-gp135 (1:50, DSHB #3F2/D8), mouse anti-HDAC7 (1:100, Santa Cruz #74563), mouse i C el ls w ith p rim ar y ci liu m [% ] ✱ Arl13b ac-Tubulin w ild ty pe w ild ty pe re sc ue G Merge + DAPI ∆P AT J Arl13b ac-Tubulin Merge + DAPI Arl13b ac-Tubulin Merge + DAPI D E F PATJ Crb3b ac-Tubulin Merge w ild ty pe ∆P AT J PATJ Crb3b ac-Tubulin Merge w ild ty pe re sc ue PATJ Crb3b ac-Tubulin Merge A B C PATJ inhibits histone deacetylase 7 to control tight junction formation and cell polarity 1 3 Page 5 of 15 333 anti-Pals1 (1:100, Santa Cruz #365411), rabbit anti-Pals1 (1:100, Proteintech #17710-1AP), rabbit anti-PATJ (1:100, SIGMA #SAB2700561), mouse anti-Occludin (1:100, Santa Cruz #271842), mouse anti-acytelated-tubulin (1:1.000, SIGMA #T6793), rat anti-ZO1 (1:100, Santa Cruz #33725).

Techniques: Staining

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 3. QKI-6 is implicated in HDAC7 signalling and promotes its splicing. (A,B) QKI-6 was overexpressed by plasmid transfection on day 1 of SMC differentiation and cells were harvested 48 h later. QKI-6 overexpression was shown to significantly induce expression of HDAC7 along with members of the downstream SMC signalling pathway (SRF and myocardin) at the mRNA and protein level. (C) Knockdown of QKI on day 3 of differentiation for 72 h suppressed SRF expression significantly, as shown by qRT- PCR. (D) Levels of HDAC7 and its splicing were observed throughout miPS-SMC differentiation. HDAC7 splicing increased, showing a significant increase in expression at day 6 of differentiation in comparison to splicing at day 2 miPS- SMCs as revealed by RT-qPCR. Levels of total HDAC7 were not significantly changed. (E) miPS-SMCs between day 2 and day 4 of differentiation were screened for the induction of a range of relevant splicing factors, including QKI-5 and QKI-6. qRT-PCR revealed only QKI-6 was significantly induced at day 4 of differentiation in comparison to day 2 differentiated cells. Data are mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Plasmid Preparation, Transfection, Over Expression, Expressing, Knockdown, Quantitative RT-PCR, Comparison

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 4. QKI-6 acts upstream of HDAC7 and directs its splicing. (A) Conventional PCR was carried out from cDNA of cell lysates overexpressing QKI-6 with primers designed to detect levels of HDAC7 splicing (HDAC7s; 314 base pairs) versus unspliced HDAC7 (HDAC7u; 371 base pairs). Overexpression of QKI-6 leads to increases in both spliced and unspliced HDAC7. (B) Effect of QKI-6 overexpression on the level of spliced HDAC7 mRNA was quantified by qRT-PCR. (C,D) Knockdown of QKI on day 3 of SMC differentiation for 72 h led to a decrease in levels of spliced HDAC7 (C); this is quantified by qRT-PCR (D). (E) To verify the QKI-6- and HDAC7-mediated mechanism of SMC differentiation and to confirm that QKI-6 lies upstream of HDAC7, HDAC7 was knocked down in day 5 miPS-SMCs, followed by QKI-6 overexpression on day 6 alongside relevant combinations of Ex-mCherry and NT controls. Cells were harvested after 48 h and qRT-PCR was carried out. Upon knockdown of HDAC7, QKI-6 overexpression was no longer able to exert its effect on inducing HDAC7, HDAC7 splicing or calponin expression. (F) Overexpression of QKI-5 in day 3 differentiating miPS-SMCs led to a significant reduction in HDAC7 splicing, but was unable to induce SMC marker expression as previously shown by QKI-6 overexpression. Differentiation was also not shown to be directed to an EC lineage upon overexpression of QKI-5 since endothelial cell markers were not observed. Data are mean±s.e.m. (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Over Expression, Quantitative RT-PCR, Knockdown, Expressing, Marker

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 5. QKI-6 binds directly to HDAC7 intron 1. (A) RBPmap software revealed a conserved QKI- binding motif that was predicted to align to intron 1 of HDAC7. (B) Specific primers were designed to incorporate this binding motif and amplify a region of 249 base pairs. (C) RNA immunoprecipitation was carried out on miPS- SMCs transduced to overexpress QKI-6 on day 4 of differentiation for 48 h. Pulldown by QKI-6 antibody showed a direct binding to HDAC7 intron 1 through conventional PCR of the resultant cDNA. Image shown is representative of three independent experiments. (D) Relative band intensity of IgG versus QKI-6 pulldown was quantified using ImageJ. Data are mean±s.e.m. (n=3). ***P<0.001.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Software, Binding Assay, RNA Immunoprecipitation

Journal: Journal of cell science

Article Title: The RNA-binding protein QKI controls alternative splicing in vascular cells, producing an effective model for therapy.

doi: 10.1242/jcs.230276

Figure Lengend Snippet: Fig. 7. Molecular mechanisms of QKI-directed vascular cell differentiation. Lineage-specific growth factors dictate the expression of the QKI alternative splicing isoforms QKI-5 and QKI-6. VEGF induces the transcription factor ETS-1, promoting QKI-5 binding to STAT3, STAT3 stabilisation and phosphorylation, VEGFR2 activation and VE-cadherin stabilisation. PDGF-BB induces QKI-6 binding to intron 1 of HDAC7, leading to its splicing and promotion of the SRF– myocardin SMC differentiation cascade. Combining QKI-derived vascular cells could be used in numerous applications such as tissue engineering, drug testing and disease modelling.

Article Snippet: CD144 (STJ96234, 1:200k ICC; 1:1000, WB) and HDAC7 (STJ93484, 1:1000, WB) antibodies were purchased from St. John’s Laboratory, London, UK.

Techniques: Cell Differentiation, Expressing, Alternative Splicing, Binding Assay, Phospho-proteomics, Activation Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Further Characterization of HDAC and SIRT Gene Expression Patterns in Pancreatic Cancer and Their Relation to Disease Outcome

doi: 10.1371/journal.pone.0108520

Figure Lengend Snippet: Panc-1 cells were transfected with either Sure Silencing shRNA Plasmid for human HDAC7 (left panel) or pCDNA3-HDAC7 plasmid (right panel). Cell clones of each (SH1 CL17/SH1 CL24 and pFlag1/pFlag3, respectively) as well as control vectors (SH CTL and pCDNA3) were analyzed. Normalized relative expression of HDAC7/28S was assessed by Q RT-PCR (A), Relative expression was calculated using 28S as control gene and normalized to Panc-1 cells. HDAC7 synthesis was evaluated by Western blot (B). Proliferation of transfected Panc-1 cell clones was evaluated by monitoring their mitochondrial respiratory chain activity using MTT assay (C). Data are means ± SD of three independent experiments. Percent proliferation was calculated as follows: at 48 h : OD at 48 h - OD at 24h /OD at 48 h; at 72h : OD at 72 h- OD at 48h / OD at 72 h; at 96h OD at 96h-OD at 72h / OD at 96 h. OD, optic densitometry.

Article Snippet: A rabbit polyclonal anti-HDAC7 antibody was from Euromedex (Souffelweyersheim, France), POD-labelled anti-rabbit antibody was from Cell Signaling (Beverly, MA).

Techniques: Transfection, shRNA, Plasmid Preparation, Clone Assay, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Activity Assay, MTT Assay

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a IHC showing the levels of HDAC7 and EphA2 in the NPCs and normal nasopharyngeal mucosa (NNM). Representative IHC images are shown on the top, and statistical analysis is presented on the bottom ( p < 0.001, Chi-squared test). b Western blot showing the levels of HDAC7 and EphA2 in the additional four paired fresh biopsies of NPC and NNM. c Western blot showing the levels of HDAC7 in the five NPC cell lines and immortalized normal human nasopharynx epithelial cell line NP69. d Kaplan–Meier survival analysis for 107 NPC patients according to HDAC7 expression levels. NPC patients with high HDAC7 expression have a significantly worse overall survival and disease-free survival than those with low HDAC7 expression. The log-rank test was used to calculate P value.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a Establishment of HK1 and 5–8F cell lines with stable knockdown of HDAC7 by shRNA (shHDAC7) and their control cell lines (shNC). b–f HDAC7 knockdown inhibits NPC cell proliferation, migration and invasion in vitro. CCK-8 ( b ), plate clone formation ( c ), and EdU incorporation ( d ) assay showing the proliferation of HK1 and 5–8F cells with shHDAC7 and their control cells. e Scratch wound healing showing the migration of HK1 and 5–8F cells with shHDAC7 and their control cells. f Transwell Matrigel invasion assay showing the invasion of HK1 and 5–8F cells with shHDAC7 and their control cells. g Xenograft growth of HK1 and 5–8F cells with shHDAC7 and their control cells. (Top) The photography of xenograft tumors after 20 days subcutaneous implantation of the cells; (bottom) growth and weight of the xenograft tumors. n = 5 mice per group. Means, SDs, and statistical significance are denoted; * P < 0.05; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: shRNA, Migration, In Vitro, CCK-8 Assay, Invasion Assay

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a Western blot showing the levels of EphA2 in the shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. b–f Restoration of EphA2 expression antagonized the inhibitory effects of HDAC7 knockdown on NPC cell proliferation, and migration and invasion in vitro. CCK-8 ( b ), plate clone formation ( c ), and EdU incorporation ( d ) assay showing the proliferation of shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. e Scratch wound healing showing the migration of shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. f Transwell Matrigel invasion assay showing the invasion of shHDAC7 HK1 and 5–8F cells transfected with EphA2 expression plasmid and their control cells. Means, SDs, and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Migration, In Vitro, CCK-8 Assay, Invasion Assay

Journal: Cell Death & Disease

Article Title: HDAC7 promotes the oncogenicity of nasopharyngeal carcinoma cells by miR-4465-EphA2 signaling axis

doi: 10.1038/s41419-020-2521-1

Figure Lengend Snippet: a Western blot showing the levels of EphA2 in the shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and EphA2 siRNA (siEphA2), and their control cells. b–f The effects of miR-4465 inhibitor transfection or miR-4465 inhibitor and siEphA2 co-transfection on NPC cell proliferation, and migration and invasion. CCK-8 ( b ), plate clone formation ( c ), and EdU incorporation ( d ) assay showing the proliferation of shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and siEphA2, and their control cells. e Scratch wound healing assay showing the migration of shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and siEphA2, and their control cells. f Transwell Matrigel invasion assay showing the invasion of shHDAC7 HK1 and 5–8F cells transfected with miR-4465 inhibitor or co-transfected with miR-4465 inhibitor and siEphA2, and their control cells. g Correlation analyses of HDAC7, miR-4465, and EphA2 in 107 NPC based on their expression levels. (Left) Spearman correlation analysis showing the positive correlation between HDAC7 and EphA2. (Right) Spearman correlation analysis showing the negative correlation between miR-4465 and HDAC7, and miR-4465 and EphA2. Means, SDs, and statistical significance are denoted; ** P < 0.01; *** P < 0.001; ns no significance.

Article Snippet: Briefly, tissue sections were incubated with anti-HDAC7 antibody (D160484–0200, BBI Life Sciences; 1:50 dilution) or anti-EphA2 antibody (6997, CST; 1:200 dilution) overnight at 4 °C, and then incubated with biotinylated secondary antibody followed by avidin-biotin peroxidase complex (DAKO) at room temperature for 30 min.

Techniques: Western Blot, Transfection, Cotransfection, Migration, CCK-8 Assay, Wound Healing Assay, Invasion Assay, Expressing