Fig. 7: Inhibition of EP300 drives the stress-resistant, invasive phenotype in melanoma cells.

a Cells were probed for EP300, CREBBP and HDAC8 by immunoblot. b, c Cells were treated with vemurafenib (1 μmol/L, BRAFi), I-CBP112 (100 nmol/L, EP300i/CREBBPi) or combination for 21 days following acetic acid permeabilization. Significance was determined by a one-way ANOVA followed by a 2-tailed t test with *=p < 0.05 (EV: p = 0.0352, HDAC8: p = 0.0129). d WM164 cells were transfected with non-silencing siRNA (siNS) or EP300 siRNA (siEP300_1 or siEP300_2). Cells were probed for EP300 and CREBBP after 72 h with ImageJ quantification. Cells were treated with dabrafenib and trametinib (100 nmol/L dabrafenib, 10 nmol/L trametinib, BRAFi-MEKi) for 72 h. Apoptosis was measured by Annexin V APC staining. Significance was determined by a one-way ANOVA followed by a 2-tailed t test with *=p < 0.05 and **=p < 0.01 (siEP300_1: p value = 0.0373, siEP300_2: p value = 0.0036). e Cells were treated with I-CBP112 (100 nmol/L) for 24 h. Lysates were collected and probed for p-c-Jun and c-Jun by immunoblot with ImageJ quantification. Cells were treated with (f) 13.85 KJ/m² UV irradiation or (g) 1% O2 for 24 h followed by cell death measurement. Significance was determined by a 2-tailed student’s t test with *=p < 0.05, **=p < 0.01 and ***=p < 0.001. In (f), WM164 p value = 0.0125 and SK-MEL-28 p value = 0.0052. In g, WM164 p value = 0.0004 and SK-MEL-28 p value = 0.0001. h, i Melanoma spheroids infected with shNS or shEP300 were plated in collagen. Scale bars = 500 µm. j Invasion area was quantified using ImageJ. Significance was determined by a 2-tailed student’s t test with *=p < 0.05, and **=p < 0.01. (WM164: p value = 0.0132 and SK-MEL-28: p value = 0.0017). k–l shNS and shEP300 SK-MEL-28 cells were injected into NOD.CB17-Prkdcscid/J mice and established for 14 days. k Brain sections were stained with H&E. Scale bars in 1x images = 4 mm. Scale bars in 10x images = 300 µm. l Number of tumors and metastases per mouse were quantified. Significance was determined in 10 mice by a 2-tailed student’s t test with *=p < 0.05. (tumor #: p value = 0.0114 and mouse metastases: p value = 0.0191) Experiments were run 3 independent times with an n of 3 in (c), 4 in (d), 5 in (j), and 8 in (f) and (g). All data are presented as mean values ± SD. Source data are provided as a Source Data file.