Rapamycin suppresses activation of mTOR signaling in active myofibroblasts.

Immunofluorescent staining of kidney sections derived from UUO models on day-1 post-obstruction was performed, using anti-αSMA (green, A–D) and anti-pS6K (red, A′–D′). Images were merged (A″–D″) and co-labeled with DAPI (A′″–D′″). Representative areas in B–B′″ (white square) were magnified in C–C′″. Arrow heads (A–A′″) and asterisks (C–C′″) indicate arterioles in the kidneys Arrows indicate representative myofibroblasts expressing pS6K (C–C′″). Scale bar: 50 µm. NIH3T3 cells were cultured for 24 hours in the absence (E–E″) or presence of 20 ng/ml recombinant human TGF-β1 (F–F′″, G–G′″), with administration of vehicle (F–F′″) or rapamycin (G–G′″). The cells were stained for αSMA (green) and pS6K (red). DAPI was used to stain the nuclei. Magnification was 400×. (H–I): Representative Western blot and quantitative assessment for expression of αSMA and pS6K in NIH3T3 cells. β-actin was used in this experiment to control for equal protein loading. *P<0.05, **P<0.01 vs. control groups. #P<0.05, ##P<0.01 vs. TGF-β+vehicle groups. Error bars represent S.E.