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. 2021 Jul 22;12(8):728. doi: 10.1038/s41419-021-04011-0

Fig. 3. Afatinib upregulates TSC1, but not TSC2, to contribute to mTOR-mediated autophagy.

Fig. 3

A FaDu, HN6, and CAL-27 cells were treated with 0, 250, 500 nM rapamycin for 24 h, and then the expression of LCB and p62 was analyzed by western blot analysis. B FaDu and HN6 cells were pretreated with 500 nM rapamycin for 30 min followed by co-incubation with 2 μM afatinib for another 24 h, and then western blot analysis was performed to examine the level of LC3B. C Western blot analysis of TSC1 and TSC2 expression in FaDu, HN6, and CAL-27 cells after treatment with 0, 1, 2, and 4 μM afatinib for 24 h. The intensity of TSC1 and TSC2 was normalized to β-actin, then the fold induction (treated/untreated control) was calculated (**p < 0.01, ***p < 0.001 vs. untreated control). D Western blot analysis of TSC1 and TSC2 expression in FaDu, HN6, and CAL-27 cells after treatment with 2 μM afatinib for 0, 4, 8, 12, and 24 h. E, F FaDu and HN6 cells were transfected with TSC1 siRNA (E) or TSC2 siRNA (F). Forty-eight hours after transfection, cells were treated with 2 μM afatinib for 24 h. The level of TSC1, TSC2, p-mTOR, and LC3B was detected by western blot analysis.