Fig 1: CARM1 is a positive regulator of CCNE2 gene in NSCLC cells. (A) ChIP analysis of human CCNE2 promoter by antibodies against CARM1, H3R17me2a, H3R26me2a or IgG in NC or CARM1-silenced PC9 and HCC827 cells. Relative enrichment of CARM1, H3R17me2a and H3R26me2a marks on the promoter regions was analyzed by real-time PCR assays. The data were presented as means ± SDs of three independent experiments; **P < 0.01, #P > 0.05. (B) The luciferase activity of CCNE2 promoter reporter was significantly increased when CARM1 (100 ng, 200 ng, 500 ng and 1000 ng) was transfected into PC9 and HCC827 cells. The CCNE2 promoter reporter luciferase activity was normalized to beta-galactosidase activity. The data were shown as means ± SDs of three independent experiments; **P < 0.01. (C) The mRNA levels of CCNE2 was downregulated in CARM1-depleted PC9 and HCC827 cells by Real-time PCR assays. ß-actin was used as an internal control. The data were presented as means ± SDs of three independent experiments; **P < 0.01. (D) The protein levels of CCNE2 was downregulated in CARM1-depleted PC9 and HCC827 cells by Western blot. GAPDH was used as loading control.
Fig 2: Restoration of CCNE2 expression abrogates the proliferation inhibition caused by CARM1 knockdown. (A) The restoration of CCNE2 in CARM1-depleted PC9 and HCC827 cells was verified by Western blot. GAPDH was used as loading control. (B) Cell proliferation capacities of NC, CARM1 KD (CARM1 shRNA) and CARM1 KD + CCNE2 OE (CCNE2 overexpression)-treated PC9 and HCC827 cells were determined by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; **P < 0.01. (C) Colony-formative abilities of NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; **P < 0.01. (D) NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 cells were subcutaneously injected into the flank of nude mice. Representative images of xenograft tumors excised from mice. (E) Tumor growth curves of NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 cells in nude mice; n =5, **P < 0.01. (F) Tumor weights of NC, CARM1 KD and CARM1 KD + CCNE2 OE-treated PC9 xenograft tumors excised from mice; n =5, **P < 0.01.
Fig 3: CARM1 is upregulated in NSCLC patients and positively correlated with CCNE2 levels. (A) Representative images of IHC staining of CARM1 in 20 cases of NSCLC patients (Tumor) and their adjacent non-tumor tissues (Normal). (B) Representative images of IHC staining of CCNE2 in 20 cases of NSCLC patients (Tumor) and their adjacent non-tumor tissues (Normal). (C) H score of CARM1 expression in 20 cases of NSCLC tumor tissues and their adjacent non-tumor tissues. **P < 0.01. (D) H score of CCNE2 expression in 20 cases of NSCLC tumor tissues and their adjacent non-tumor tissues. **P < 0.01. (E) Pearson correlation analysis was performed to examine the correlation between CARM1 and CCNE2 expression in 20 cases of NSCLC patients (n = 20; r = 0.6958; P < 0.01). (F) Analysis of data from the Kaplan-Meier plotter database suggested that high expression of CARM1 (Cutoff value: 262) was associated with shorter 10-year overall survival of NSCLC (lung adenocarcinoma, n = 461) patients. P < 0.01. (G) Analysis of data from the Kaplan-Meier plotter database suggested that high expression of CCNE2 (Cutoff value: 228) was associated with shorter 10-year overall survival of NSCLC (adenocarcinoma) patients. P < 0.01.
Fig 4: Inhibition of CARM1 enzymatic activity represses CCNE2 expression in NSCLC cells. (A) ChIP analysis of human CCNE2 promoter by antibodies against CARM1, H3R17me2a, H3R26me2a or IgG in DMSO or EZM2302-treated (10 nM) PC9 and HCC827 cells. Relative enrichment of CARM1, H3R17me2a and H3R26me2a marks on the promoter regions was analyzed by real-time PCR assays. The data were presented as means ± SDs of three independent experiments; **P < 0.01, #P > 0.05. (B) The protein levels of CCNE2, H3R17me2a and H3R26me2a were downregulated in EZM2302-treated (10 nM) PC9 and HCC827 cells by Western blot. GAPDH or histone H3 were used as loading controls. (C) The mRNA levels of CCNE2 was downregulated in EZM2302-treated (10 nM) PC9 and HCC827 cells by Real-time PCR assays. β-actin was used as an internal control. The data were shown as means ± SDs of three independent experiments; **P < 0.01. (D) Cell proliferation abilities of DMSO or EZM2302-treated (10 nM) PC9 and HCC827 cells were assessed by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; **P < 0.01. (E) Colony-formative abilities of DMSO or EZM2302-treated (10 nM) PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; **P < 0.01.
Fig 5: CARM1 promotes NSCLC cell proliferation in vitro. (A) Cell proliferation abilities of CARM1-depleted PC9 and HCC827 cells were assessed by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; **P < 0.01. (B) Colony-formative abilities of CARM1-depleted PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; **P < 0.01. (C) Overexpression of CARM1 in PC9 and HCC827 cells was examined by Western blot. GAPDH was used as loading control. (D) Cell proliferative abilities of CARM1-overexpressed PC9 and HCC827 cells were assessed by CCK-8 assays. The data were presented as means ± SDs of three independent experiments; **P < 0.01. (E) Colony-formative abilities of CARM1-overexpressed PC9 and HCC827 cells were determined by colony-formation assays. Right panel, the relative colony-formative abilities (% of NC) were quantified. The data were shown as means ± SDs of three independent experiments; **P < 0.01.
Supplier Page from Abcam for Anti-CARM1 antibody