Fig 1.
MPC1 is expressed in the human hair follicle and MPC inhibition stalls cell cycle progression and disrupts the expression of hair follicle signalling pathway genes.
A) MPC1 and PDK immunoreactivity in the human hair follicle. CTS–connective tissue sheath. DP–dermal papilla; DSC–dermal sheath cup; GL–germinative layer; HS–hair shaft; IRS–inner root sheath; L-ORS–lower outer root sheath; SG–sebaceous gland. Regional analysis (zones of analysis indicated by red dashed lines) performed on 7 anagen hair follicles from 3 donors. Mann Whitney test, p value *** 0.0006. Scale bar 50 μm. B) Fluorescent EdU labelling on human hair follicle tissue sections shows how UK-5099 treatment blocks DNA replication in the hair follicle both within the bulge epithelium and hair matrix (HM). DP—dermal papilla. Scale bar 50 μm. C) Quantitative analysis of EdU and Ki-67 in the bulge and hair matrix following UK-5099 treatment. Ordinary One-way Anova with Multiple Comparisons. EdU analyses: Adjusted p-values *** 0.0002, **** <0.0001. Ki-67 analyses: Adjusted p-values ** 0.0018; *** 0.0006; **** <0.0001. N = 2–3 donors (6–10 independent anagen hair follicles per condition). Plotted line is the mean. D) Dot plot of the top 10 enriched IPA pathways following 40 μM UK-5099 treatment. Analysis conducted on 1206 genes with 2-fold change and padj <0.05. See also S3 Fig in S1 File. E) Volcano plot annotated with differentially expressed genes involved with FGF, IGF, TGFβ and Wnt signalling with an adjusted p value < 0.05 following treatment of human hair follicles with 40 μM UK-5099.
Fig 2.
MPC inhibition promotes mitochondrial dysfunction in the human hair follicle.
A) Volcano plot annotated with differentially expressed genes involved with mitochondrial function with an adjusted p value < 0.05 following treatment of human hair follicles with 40 μM UK-5099. B) Staining with the mitochondrial marker TOMM20 comparing vehicle and 40 μM UK-5099 treated human hair follicles. Analysis of intensity (FI) and TOMM20+ aggregates reveals changes in the hair matrix following MPC inhibition. Mann-Whitney test. Exact p-values **** <0.0001. N = 5 donors (16 independent anagen hair follicles per condition). Scale bar 50 μm.
Fig 3.
MPC inhibition activates the integrated stress response in the human hair follicle.
A) Dot plot of the top enriched pathways for upregulated and downregulated genes, following 40 μM UK-5099 treatment generated using ReactomePA. Analysis was conducted by filtering the top upregulated and downregulated genes (adjusted p value < 0.05 and a 2-fold change), analysing 117 and 490 genes respectively. ReactomePA analysis was performed in R [34]. Results were adjusted for multiple testing using the Benjamini and Hochberg method. B) Heatmap of upregulated genes (from top 100 genes by adjusted p value, see S4 Fig in S1 File for heatmap of top differentially expressed genes) in the human hair follicle following 40 μM treatment with the MPC inhibitor UK-5099. Genes reported to be targets of ATF4 are annotated in green. C) Volcano plot with annotated genes identified to be regulated by ATF4 in IPA. 33 annotated ATF4 target genes identified from list of 1206 genes with an adjusted p value < 0.05 and a 2-fold change following treatment of human hair follicles with 40 μM UK-5099. D) Images of ADM2 and ATF4 (mRNA FISH), and Ki-67 expression in the hair matrix in hair follicles treated with UK-5099 or UK-5099 + ISRIB (ISR inhibitor). Scale bar 50 μm. Quantitative analysis of ADM2, ATF4, Ki-67 and Cleaved Caspase 3 (CC3) comparing UK-5099 + ISRIB treatment versus UK-5099 alone and/or vehicle (see also S8-S10 Figs in S1 File). Ordinary One-way Anova with Multiple Comparisons. Adjusted p-value **** <0.0001. Plotted line is the mean. N = 4–6 donors (16–22 independent anagen hair follicles per condition).