Fig. 3

GABAergic signaling drives melanoma/keratinocyte communication. A, Schematic representation of the LOPAC small-molecule library screen in human melanoma/keratinocyte cocultures treated with control (DMSO) or LOPAC 1280 library compounds (10 ?mol/L each, indicated by their Sigma library identifiers) for 48 hours and quantified for an increase in switching efficiency (created with BioRender.com). B, Fold change over control (DMSO) in switching efficiency in the top 28 hits of the LOPAC small-molecule library screen. Red bars indicate compounds that are agonists or allosteric modulators of the GABA-A receptor; blue bar represents DMSO control. C, Percentage switching efficiency calculated as number of switched cells per well normalized to control (DMSO) upon treatment with the GABA antagonist picrotoxin (100 ?mol/L, 1 mmol/L, 10 mmol/L) in melanoma/keratinocyte cocultures for 48 hours pooled from 6 biological replicates (n = 12; ratio: 1:3, keratinocyte:melanoma). Error bars, SD; P values generated by one-way ANOVA with multiple comparisons; ****, P < 0.0001. D, Schematic representation of the F0 zebrafish genetic reporter assay to quantify changes in keratinocyte switching efficiency in zebrafish embryos treated with a GABA-A agonist (muscimol) or a GABA-A antagonist (picrotoxin). Created with BioRender.com. E and F, Percentage switching efficiency calculated as percentage of RFP-positive area normalized to GFP-positive area, normalized to control in 3 days post-fertilization zebrafish embryos treated with muscimol (10 ?mol/L; E) or picrotoxin (100 ?mol/L; F). Data represent n = 44 DMSO-treated fish, n = 42 muscimol-treated fish, and n = 44 picrotoxin-treated fish pooled from 3 biological replicates. Error bars, SD; P values generated by two-tailed unpaired t test; ****, P < 0.0001. G, Waterfall plot of enriched pathways from GSEA of switched vs. parental keratinocytes. GABA-A receptor pathways are highlighted in red. H, Immunostaining for GAD1 (enzyme) and GABA in A375 melanoma cells. Individual cells are pseudocolored as indicated. I, Schematic representation of the human in vitro switch reporter assay in melanoma/keratinocyte cocultures with genetic loss of function in GABA pathway components. Created with BioRender.com. J, Percentage switching efficiency calculated as number of switched cells per well normalized to control siRNA when cocultures were treated with a combination of GAD1/2 (melanoma cells) and ARHGEF9 (keratinocytes) targeting siRNA pooled from 3 biological replicates (n = 12; ratio: 1:3, keratinocyte:melanoma). Error bars: SD; P values generated by two-tailed unpaired t test; ****, P < 0.0001.