Fig. 8: Inhibition of ERBB signaling blocks MCL‑1 protein expression and hair regeneration.

A Kinase inhibitor screen in primary keratinocytes identifies pathways that modulate MCL‑1 protein levels. Cells from newborn FVB/N pups were treated with 2 µM of each inhibitor from a panel of 154 kinase inhibitors for 24 h, followed by Western blotting for MCL‑1 expression. The intensities of MCL‑1 and ACTIN were analysed using Image J. The ratio of MCL‑1 vs Actin in inhibitor‑treated cells with normalization to DMSO treated control cells were plotted as a bar graph. Inhibitors were grouped based on the kinase targeted. Groups highlighted in blue show more than a 50% reduction of MCL‑1 levels on average (MCL‑1 vs Actin ≤ 0.5). B Validation of ERBB inhibitors from (A) shows significant reduction in MCL‑1 protein levels after 24 h of treatment (n = 2 independent experiments). C Western blot of keratinocytes treated with various afatinib concentrations for 24 h demonstrates MCL‑1 reduction without affecting other BCL‑2 family proteins (n = 3 independent experiments). D Afatinib inhibits phosphorylation of protein translation regulators in primary keratinocytes. Western blot of p‑EGFR, p‑ERBB2, p‑P70 S6K, p‑S6, p‑eIF4B, and p‑eEF2K (n = 2 independent experiments). E qPCR analysis of afatinib‑treated keratinocytes reveals Mcl‑1 transcript levels remain stable. Data are mean ± SEM (n = 3 independent experiments). F H&E‑stained sections of dorsal skin from wildtype FVB/N mice treated with 50 mg/kg afatinib or vehicle followed by depilation show impaired hair regeneration at 9 dpd (n = 9 mice per treatment group). Scale bars, 50 µm. G, H FACS plots and bar graphs indicate changes in CD34 and SCA‑1 populations in CD49f⁺ epithelial cells from afatinib‑ or vehicle‑treated mice at 9 dpd. Data are mean ± SEM. ****p < 0.0001; **p < 0.01; *p < 0.05; Student’s t‑test. I–K Confocal images of HFs from afatinib‑ or vehicle‑treated mice at 9 dpd. I MCL‑1 and p‑ERBB2 expression; J MCL‑1 and p‑S6 expression; K Proliferating (PCNA⁺) and apoptotic (CC3⁺) cells. Scale bars, 50 µm. Basal layer stained by KRT14 and DAPI as nuclear counterstain (n = 6 mice per treatment arm).