Figure 2(A) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 23-30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (B) Representative images of p-S6 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Nuclei stained with DAPI.(C) Hair cycle staging was performed using Ki-67 and Masson-Fontana histochemistry Mean ± SEM; N = 36-39 HFs per group from 6 different donors treated with Rapamycin 20 ng/ml or vehicle (control) for 7 days. (D) Representative fluorescence images of Ki-67 and bright field microscopic images of Masson-Fontana. for C-D where all HFs were analyzed) were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(E) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 28-30 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (F) Representative bright field microscopic images of Masson-Fontana histochemistry. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test(G) Quantitative analysis of tyrosinase activity. N = 18-21 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (H) Representative images of tyrosinase activity immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(I) Quantitative analysis of gp100 expression. N = 24-26 anagen VI HFs from 8 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (J) Representative images of gp100 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(K) Quantitative analysis of melanocyte dendricity. N = 22-24 anagen VI HFs from 6 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (L) Representative images of gp100 immunofluorescence. Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. Mean ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(M) Quantitative analysis of α-MSH expression. N = 17-20 anagen VI HFs from 5 different donors treated with Rapamycin 20 ng/ml or untreated (vehicle) for 7 days. (N) Representative images of α-MSH immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. ± SEM, Unpaired Student's t-test Nuclei stained with DAPI.(O) Quantitative analysis of phosphorylated S6 (p-S6) (mTORC1 activity read-out) immunoreactivity. N = 8-10 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng/ml, 2ug/ml Agouti, Rapamycin+Agouti or untreated (vehicle) for 7 days. (P) Representative images of p-S6 immunofluorescence. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and One-Way ANOVA Nuclei stained with DAPI.(Q) Quantitative histomorphometry of melanin production by Masson-Fontana histochemistry. N = 8-11 anagen VI HFs from 2 different donors treated with Rapamycin 20 ng/ml, 2 ug/ml Agouti, Rapamycin+Agouti or untreated (vehicle) for 7 days. (R) Representative bright field microscopic images of Masson-Fontana histochemistry. : Only anagen VI HFs were investigated and analyses performed in defined reference areas (dotted areas) in the HFPU. , * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and One-Way ANOVA