(A–D) Highly pigmented iHPMs were infected with lentiviruses expressing TSC1 shRNA or scrambled control (Scr) shRNA and selected with puromycin. (A) Immunoblot analysis shows that TSC1 depletion led to reduced MITF-M and its downstream targets PGC-1α and TYR, as well as mTORC1 activation. (B) Cell pellets from TSC1-depleted cells had reduced pigmentation. (C and D) TSC1-depleted cells had reduced melanin and TYR activity. (E–H) Entirely similar to the data shown in A–D, except that TSC2 shRNAs were expressed. (I–L) TSC2-depleted iHPMs were reconstituted with lentiviruses containing WT TSC2 or patient-derived TSC2-mutant (P419S) or empty vector control viruses (EV) and selected with puromycin. (I) Immunoblot analysis shows that WT TSC2, but not the TSC2 mutant, rescued mTORC1 activation, MITF and PGC-1α expression, and TYR protein levels. (J) Pigmentation, (K) melanin content, and (L) TYR activity were also all rescued by WT TSC2. Data in C, D, G, H, K, L, and M represent the mean ± SD from at least 3 independent experiments. *P < 0.05, by 2-tailed Student’s t test. (M) Expression of the indicated genes was measured by qPCR in control or shRNA-expressing cells. GAPDH was used as a control. (N) Immunoblot analysis of lysates from TSC1- or TSC2-depleted cells treated with MG132 (25 μM) for 6 hours. MITF, PGC-1α, and TYR were not rescued by MG132 treatment. (O and P) TSC1- or TSC2-depleted cells were infected with lentiviruses containing MITF-M or empty vector control (Scr) and selected with puromycin. (O) Immunoblot analysis shows increased MITF and TYR expression in cells infected with MTIF-M lentivirus. (P) Ectopic expression of MITF rescued the pigmentation loss in TSC1- and TSC2-depleted cells. The immunoblots presented in the figure panels include replicate samples run on parallel gels.