(A) Normalized read counts for MIR328-3p (top) and MIR211-5p (bottom) in TPA-containing (+TPA) or TPA-free media, with or without 15.6 ng/ml doxycycline (Dox). See Supplementary file 5 for individual values and exact p values. (B) Volcano plots depicting log2 fold change against adjusted p value (−log10) from differential expression (DE) analysis of small RNA sequencing of indicated comparisons. MIR211-5p labeled and shown in red. MIR328-3p shown in blue. (C) First and second principal components (PCs) separating diBRAFV600E melanocytes cultured in indicated conditions. (D) Volcano plots depicting log2 fold change against adjusted p value (−log10) from DE analysis of mRNA sequencing of all +TPA specimens versus all TPA-free specimens (top) or all Dox specimens versus all no-Dox specimens (bottom). (E) Normalized read counts for AURKB normalized to no Dox conditions (n=3). P values from unpaired t-tests. Sequencing data represented in (A–F) are from n=3 per condition analyzed with DESeq2. (F) Gene set enrichment analysis (GSEA) comparing DE gene sets from (D) to Molecular Signature Database Hallmark gene sets, KEGG Pathway gene sets and published signatures of melanocyte signaling and differentiation from Belote et al., 2021; Tsoi et al., 2018; Tirosh et al., 2016; Ryu et al., 2011; Hoek and Goding, 2010. All enrichments with adjusted p value<0.025 are depicted. See Supplementary file 6 for all associations. Normalized enrichment score (NES). Enrichment differences between pathways across the four conditions in (C) are depicted in Figure 4—figure supplement 1. (G) Representative photomicrographs of immunofluorescence for MLANA or AXL in human melanocytes cultured in +TPA or TPA-free conditions. White scale bar=30 µm. (H) Quantification of fluorescence intensity (arbitrary pixel intensity units, a.u.) from experiments represented in (G). Box (median, 25th and 75th percentiles) and whiskers (10th and 90th percentiles) of 84, 88, 113, and 96 cells, respectively. P values from unpaired t-tests. (I) Representative plots (top) and quantification (bottom, n=4) of relative aldehyde dehydrogenase activity of melanocytes grown in +TPA or TPA-free conditions. Normalized read counts for AURKB normalized to no Dox conditions (n=3). P values from unpaired t-tests. Sequencing data represented in (A–G) from n=3 per condition analyzed with DESeq2. (J) Schematic of model supported by transcriptomic analyses. TPA induces a more differentiated state in human melanocytes. Due to increased MIR211-5p and base-line MIR328-3p expression, AURKB expression is capped, resulting in G2/M accumulation upon BRAFV600E expression. (K) Design of pLVX vectors expressing microRNA sponges fused to zsGreen. (L) Mean and standard deviation for cell number as percent of time point 0 hr after 7 days of culture in +TPA media with or without 15.6 ng/ml Dox (n=3 per condition). Melanocytes were transduced with either pLVX-control, -AURKB, -anti-MIR211-5p, or -anti-MIR328-3p. P values from unpaired t-tests compared to matched no Dox condition (horizontal), Control no Dox condition (vertical in green bar), or Control Dox condition (vertical in red bar). TPA, tetradecanoylphorbol acetate.