Fig. 5. Impact of LZTR1 knockdown on apoptosis and pigmentation.

a Gene set enrichment analysis (GSEA)³⁴ showing concordance in hallmark pathways among bulk acral melanoma tumors (same as Supplementary Fig. 5e), acral melanoma single-cell transcriptomes (scRNA-seq; same as Supplementary Fig. 5e), and a primary melanoma cell line (LZTR1 vs. KD), in relation to high vs. low LZTR1 expression. All pairwise comparisons are statistically significant by Spearman correlation (nominal P ≤ 0.0005) except for “scRNA-seq” vs. “LZTR1 vs. KD”. Gold, positive normalized enrichment score (NES); blue, negative NES; KD knockdown. b LZTR1 shRNA and sgRNA (CRISPR-Cas9) induce apoptosis in melanoma cells. Data were expressed as the average of triplicate or duplicate wells with error bars indicating 95% confidence intervals (CI). c, d Effects of inhibiting caspase activity with IDN-6556 (IDN, 2 µM, 3 days). As shown in c, IDN-6556 suppressed shLZTR1-induced caspase activity. Data in c are expressed as the average of triplicate (shRNA only and shRNA + IDN-6556), or duplicate (control) wells with error bars denoting 95% CI. As shown in d, IDN-6556 increased the levels of LZTR1 (known to be degraded by caspases) and rescued caspase substrates, such as GOLGA4, p53, and to a lesser extent, NF-κB. e Effect of shLZTR1 on cell morphology and actin filament organization. Actin filaments were visualized by staining with rhodamine-phalloidin (magenta) and the Golgi with anti-GM130 (green, Cy2). The nuclei are stained with DAPI (blue). Scale bar = 50 µm. f–h Impact of shLZTR1 on MITF (panel f), tyrosinase (TYR) (panel g), and pigmentation (panel h). shRNAs in b–d and f–h are indicated by unique numerical identifiers. C scrambled shRNA control. Actin levels in d, f and g show protein loading. Cell lines are indicated above all plots in b–h and colored according to their origin: acral melanoma (blue), sun-exposed melanoma (red). Panel e represents one of two experiments. Source data are provided as a Source Data file.