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. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: Cancer Discov. 2020 Apr 8;10(7):980–997. doi: 10.1158/2159-8290.CD-19-0532

Figure 2. G9a is required for growth in G9a copy number-gained melanoma cells.

Figure 2.

(A) Proportions of TCGA melanomas with different G9a copy numbers. (B) Representative western blot of H3K9me2 in melanoma cell lines and primary human melanocytes. The numbers indicate qPCR-determined G9a copy numbers in G9a-gained/amplified (red) and -unamplified (black) melanoma lines and melanocytes; ND, not determined). The experiment was repeated four times independently (refer to Supplementary Fig. S3F). (C) Colony formation of G9a-gained or -amplified/H3K9me2-high and H3K9me2-low melanoma cell lines with G9a knockdown or control shRNA (shLuc). Data represent mean +/− SD of triplicates. P-values were calculated by one-way ANOVA with the Holm-Šidák correction for multiple pairwise comparisons. *p<0.05, **p<0.01, ****p<0.0001 vs. shLuc. (D) Dose-dependent growth inhibition by UNC0638 in melanoma cell lines and primary human melanocytes. Data represent mean +/− SD of triplicates from at least two independent experiments. (E) Western blotting for autophagy marker LC3B in G9a-gained melanoma cell lines WM983B and K029, and G9a-unamplified melanoma cell line UACC62. Cells were treated with UNC0638 at the indicated concentrations for 72h. Representative images from two-independent experiments were shown. (F) In vivo effect of potent G9a/GLP inhibitor UNC0642 (2.5 mg/kg) on growth of xenografts from G9a-gained/H3K9me2-high melanoma cell line K029. *p<0.05, ***p<0.001 by repeated measures two-way ANOVA with the Holm-Šidák correction for multiple pairwise comparisons of the two groups at each time point. N=5/group. (G) Representative immunohistochemistry images (40X) of H3K9me2 (top) and LC3B (bottom) in K029 melanoma xenograft tissue samples from mice treated with vehicle or UNC0642. (H) Growth of individual WM983B xenograft tumors in mice treated daily with vehicle (10% DMSO/PBS) (left, n=9) or UNC0642 (2.5 mg/kg) (right, n=10). Red lines (2/10) indicate complete tumor regressions. (I) Kaplan-Meier survival curves of WM983B-xenografted mice treated with vehicle or UNC0642 (2.5 mg/kg). **p<0.01 by the log-rank (Mantel-Cox) test.