Fig 1. mTOR signaling is deactivated during differentiation and only partially contributes to the control of proliferation.

(a) Increasing densities of HaCaT were seeded to promote differentiation and harvested after 72h. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies. (b) NHK were serum-starved and differentiation was induced with 2mM CaCl₂ for the indicated time points. Protein lysates were subjected to SDS-PAGE and Western Blotting with the indicated antibodies was performed. Below a densiometrical quantification of involucrin and P-S6 levels of n = 4–5 similar blots is shown. Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (* p ≤0.05, ***p ≤0.001). (c) Keratinocytes stem cells (KSC), transient amplifying (TA) and postmitotic (PM) cells were separated according to their ability to adhere to type IV collagen. Protein lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies, showing that mTORC1 signaling is mainly present in undifferentiated cells. (d) Normal human skin was stained with P-mTOR S2448 and Ki-67 antibody. Nuclei were stained with DAPI. Single color and overlay images are presented, which show that mTOR is activated in proliferation cells of the basal layer. Bars represent 100 μm. (e) HaCaT cells were reverse-transfected with siRNA targeting Akt, Raptor or control siRNA and seeded in 96 well plates. After 48h proliferation was quantified using the XTT-based assay. Graph presents mean ± SEM (n = 4–8). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (f) To control for the efficiency of knockdown, HaCaT cells were transfected as in (e) and seeded in 6 well plates. Protein lysates were harvested after 48h and a Western blot was performed with the indicated antibodies. To show the consequences of Akt and Raptor knockdown, phosphorylation of the downstream targets GSK-3 and S6 was also captured. (g) HaCaT cells were seeded in 96 well-plates in triplicates and after 24h 50 μM LY294002, 100 nM Rapamycin or 250 nM Torin or solvent (DMSO) were added. After another 48h cell proliferation was measured with a BrdU assay. Graph presents mean ± SEM (n = 6). Statistical significance was calculated with one-way ANOVA and Bonferroni multiple comparison (****p ≤0.0001, ns: non-significant). (h) To show efficiency of the used inhibitors, HaCatT cells were treated as in (g). Protein lysates were prepared and a Western blot was performed with the indicated antibodies.