Fig. 6.

β-TrCP1 and β-TrCP2 regulate autophagy and growth in different manners via modulating mTORC1. SK-BR3 and MDA-MB-231 cells stably expressing GFP-LC3 were transfected with siRNA targeting β-TrCP1, β-TrCP2, or β-TrCP1 + 2 or with a scrambled control siRNA for 48 h. Cells were then photographed under a fluorescence microscope (a, top) and subjected to IB (b) with anti-LC3, p62, p-S6K1, t-S6K1, p-S6, t-S6, β-TrCP1, β-TrCP2, and Actin Abs. Cells with punctate structures of GFP-LC3 were counted, and their number was expressed as percentage of autophagy (a, bottom) (mean ± S.E.M., n = 5, ***p < 0.001, NS, not significant, compared with cells transfected with scramble control siRNA oligos). SK-BR3 and MDA-MB-231 cells were transfected with the indicated siRNA for 48 h and then subjected to an ATPlite cell growth assay (mean ± S.E.M., n = 3, *p < 0.05, **p < 0.01, compared with cells transfected with scramble control siRNA oligos) (c) or IB with anti-p-S6K1, t-S6K1, p-S6, t-S6, β-TrCP1, β-TrCP2, and Actin Abs (d). The band density was quantified and expressed as fold change compared with the corresponding control by setting the control value as 1 (b, d)