CDK4/6 inhibition antagonises paclitaxel cytotoxicity in the stem/progenitor cell-rich human hair follicle outer root sheathA) Schematic of experimental design.Figure 7A) Schematic of experimental design. Hair follicles (HFs) were pre-treated with (or without) palbociclib for 18h then further incubated either with/without paclitaxel for a further 24h. Subsequently, drug-containing media was washed out and HFs were cultured for an additional 24-48h period. B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error C) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error D) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error E) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. ; wo - washout. 50 µm scale; F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 50 µm scale Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale. Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.