Fig 2: BAP18 significantly regulates the transcription of a series of genes in OSCC-derived cell lines. (a) The effects of BAP18 depletion on a series of genes in Cal-27 cells by real-time quantitative PCR. Three independent siRNAs against BAP18 (siBAP18s) were designed and generated for BAP18 depletion. Levels of all kinds of RNAs as indicated were normalized to that of β-Actin. Student t-test were used, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05. (b) Effect of knockdown of BAP18 on mRNA expression of CCND1, CCND2, or CCND3 in SCC9 cells. Student t-test were performed, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05. (c,d) Effects of depletion of BAP18 or ectopic expression of BAP18 on protein expression of cyclin D1 and cyclin D2 by western blotting in Cal-27 cells (c) or SCC9 cells (d) with indicated antibodies.

Fig 3: BAP18 is highly expressed in clinical OSCC samples. (a) Expression of BAP18 in unpaired fresh tissues of oral non-cancerous (NC) tissues and OSCC tissues. (b) Grayscale values of western blotting in (a), Student t-test was used for statistical significance between two groups. (c) BAP18 expression in non-cancerous (NC) tissues and OSCC samples with different clinical stages. Magnification: 4* and 20*; Scale bars: 500 μm and 100 μm. (d) Statistical significance among non-cancerous tissues, early stages tissues (I and II) and terminal stages tissues (III and IV). Average score of three independent view calculation represents the sample final score. Mann-Whitney U test were used, ***p<0.001. (e) Ki67 and BAP18 expression in OSCC samples. Magnification: 10*; Scale bars: 100 μm. (f) Scatter diagram of two proteins (BAP18 and Ki67) expression in the same sample, and the straight line was generated by Prism Graphpad7.0. (g) Disease free survival curves was generated from GEPIA showing BAP18 contribution to HNCC patients living from TCGA cohorts (http://gepia.cancer-pku.cn/index.html).

Fig 4: BAP18 depletion influences the G1-S phase in OSCC-derived cell lines. (a) Cal-27 cells were infected with lentivirus against BAP18 (shBAP18) and its negative control (shCtrl). The efficiency of the shBAP18 detected by western blotting in Cal-27 cells. (b–d) The impact of BAP18 on cell cycle progression by flow cytometry assay in Cal-27 cells. (e) SCC9 cells were infected with lentivirus against BAP18 (shBAP18) and its negative control (shCtrl). The efficiency of the shBAP18 detected by western blotting in SCC9 cells. (f–h) The impact of BAP18 on cell cycle progression by flow cytometry assay in SCC9 cells. All statistics were performed by Graphpad7.0 with Student t-test, ns stands for no significant, ***p<0.001, **p<0.01, *p<0.05.

Fig 5: BAP18 associates with ERα to promote ERα-mediated transactivation. (A) HEK293 cells were co-transfected with PcDNA3.1/FLAG-BAP18 and ERα plasmids with or without estrogen (E2, 100 nM). Whole-cell extracts were immunoprecipitated with the anti-FLAG antibody or IgG after transfection for two days. Input means 5% of the whole extract for each column. (B) HEK293 cells were co-transfected with ERα and truncated mutant of BAP18 expression plasmids. All BAP18 expression plasmids carried GFP-tagged and vGFP plasmids were used for the control group. The location of the star indicates where the protein is expressed. BAP18-FL meant BAP18 full-length expression plasmid. BAP18-N, BAP18-C represented N-terminus of BAP18 expression plasmid and C-terminus of BAP18 expression plasmid. (C, D) Co-IP experiments showing the interaction between endogenous BAP18 and ERα in MCF7 cells. (E) The distribution of BAP18 and ERα with or without E2 by immunofluorescence staining in MCF7 cells. The cells were stained with anti-DAPI (Blue), anti-BAP18 (Green), and anti-ERα (Red). Scale bars were 50um. (F) BAP18 enhances ERα-induced transactivation in HEK293 cells. The cells were co-transfected with ERα expression plasmid together with FLAG-tagged BAP18 expression plasmid (FLAG-BAP18) or PcDNA3.1 plasmid with (white histograms) or without (gray histograms) E2 treatment (100nM). The expression levels of FLAG-BAP18 or ERα were detected with anti-FLAG and anti-ERα by western blotting. (G) MCF7 cells were co-transfected with full-length BAP18 or its truncated mutants as indicated. The expression levels of BAP18 and its truncated mutants were examined with anti-GFP by western blotting. (H) BAP18 increases ERα or ERα AF-2 mediated transcriptional activity. The expression plasmids of ERα full length or truncated mutants carrying ERα AF-1 or ERα AF-2 were transfected into HEK293 cells with or without BAP18 expression plasmid as indicated. Relative luciferase activity as shown above is the mean value at least three times. Student t-tests were used and error bars represent mean ± SD. *P< 0.05, **P< 0.01 and ns stands for no significance.