Fig. 1: HDAC8 expression confers stress resistance in melanocytes and melanoma. | Nature Communications

Fig. 1: HDAC8 expression confers stress resistance in melanocytes and melanoma.

From: HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis

Fig. 1

a Cell lines treated with vemurafenib (BRAFi) were organized into BRAFi sensitive (IC50 < 1 µM) BRAFi intermediate (IC50 = 1), and BRAFi resistant (IC50 > 1 µM). Cells were probed for HDAC8, acetylated SMC3 (acSMC3), SMC3, phospho-EGFR (p-EGFR), EGFR, phospho-c-Jun (p-c-Jun), c-Jun, and MITF by immunoblot. b Cells transfected with an empty vector (EV) or HDAC8 construct were probed for HDAC8, acSMC3, SMC3, p-c-Jun, c-Jun, and EGFR by immunoblot with ImageJ quantification. c Cells were treated with vehicle (1:1000 DMSO, VC), or with BRAF and MEK inhibition (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 (WM164: p = 0.0148 and SK-MEL-28: p = 0.0104). Cells were treated with (d) 13.85 KJ/m2 UV irradiation or (e) 1% O2 for 24 h followed by cell death measurement by trypan exclusion. Significance was determined by a one-way ANOVA followed by a 2-tailed t test with ***=p < 0.001 and **=p < 0.01. In (d), WM164 p value < 0.0001 and SK-MEL-28 p value < 0.0001. In (e), WM164 p value = 0.0016 and SK-MEL-28 p value = 0.0014. f Primary melanocyte cells were treated with 13.85 KJ/m2 UV irradiation for indicated time points. Lysates were collected and probed for p-c-Jun, c-Jun, HDAC1, HDAC2, HDAC3, and HDAC8 by immunoblot with ImageJ quantification. g HERMES1 and 3 cells (melanocytes) were transfected with an EV or HDAC8 construct and probed for HDAC8, MITF, p-c-Jun, and c-Jun by immunoblot with ImageJ quantification. HERMES1 and 3 cells were treated with (h) 13.85 KJ/m2 UV irradiation or (i) 1% O2 for 24 h followed by cell death measurement by trypan exclusion. Significance was determined by a one-way ANOVA followed by a 2-tailed t test with *=p < 0.05 and **=p < 0.01, and ***=p < 0.001. In (h), HERMES1 p value = 0.028 and HERMES3 p value = 0.0008. In (i), HERMES1 p value = 0.0198 and HERMES3 p value = 0.0063. All experiments were run 3 independent times with an n of 3 in each cohort in (c) and an n of 8 in each cohort in (d), (e), (h) and (i). All data are presented as mean values ±SD. Source data are provided as a Source Data file.

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