GASC1 Promotes Stemness of Esophageal Squamous Cell Carcinoma via NOTCH1 Promoter Demethylation

Figure 6

Blockade of GASC1 induces NOTCH1 promoter methylation. (a-c) ChIP analysis of H3K9 methylation levels at NOTCH1 promoter region after GASC1 knockdown (shGASC1) in ALDH⁺ KYSE150 cells was quantified by qPCR. Relative promoter occupancies (% input) are shown with error bars based on standard errors calculated from at least three replicates. The input signal is set as 100% (not depicted in graphs) for each set of assays. Specific antibodies that individually recognize either the di- (H3K9me2) or trimethylated form of H3K9 (H3K9me3) were used. GST antibody was used as a control. (d-e) ChIP analysis of H3K9me2 and H3K9me3 levels at the NOTCH1 promoter after CA treatment (2, 5, 10, and 20μM) in ALDH⁺ KYSE150 cells was quantified by qPCR. ALDH⁺ KYSE150 cells before and after GASC1 knockdown and treatment with CA (10 μM) subjected to double immunofluorescence for GASC1 (green), H3K9me2 (g) / H3K9me3 (h) (red), and DAPI (blue). One representative micrograph is shown. Scale bar represents 30 μm. Data are represented as means ± SD. = P < 0.05; ns = nonsignificant.

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