Figure 3.

c-Abl Regulates TFEB Independent of mTORC1 Activity

(A) Western blots of endogenous TFEB in HeLa cells treated with imatinib 10μM for 3 h. Torin1 0.3μM and starvation media (STV) for 3 h were used as a positive control.

(B) Representative Western blot and quantification of TFEB phosphorylated on S142 normalized against GAPDH in HeLa TFEB-GFP cells treated with imatinib 10μM for 3 h and siRNA c-Abl for 48 hr. STV media for 3 h was used as positive control. n = 3 independent experiments.

(C) Representative Western blot and quantification using the 14-3-3 antibody that binds to phosphorylated TFEB on S211. For immunoprecipitated GFP from HeLa TFEB-GFP, cells treated with imatinib 10 μM and Torin1 0.3 μM for 3 h. n = 3 independent experiments.

(D) Representative Western blot and quantification of phospho p70-S6K normalized against GAPDH in HeLa cells treated with imatinib and nilotinib 10μM for 3 h. Torin1 0.3μM and STV media treatment for 3 h were used as positive controls. n = 3 independent experiments. Scale bars, 10 μM.

(E) Representative confocal microscopy images and quantification of TSC2 KO cells transfected with the TFEB-GFP plasmid. Cells were treated with imatinib 10μM for 3 h and with Torin1 0.3μM for 3 h as a positive control. n = 40 cells per conditions. Scale bars, 10 μM.

(F) Representative images and quantification of percentage of nuclear TFEB-GFP in HeLa TFEB-GFP cells synchronized with STV media for 1 h. Then, cells were treated with imatinib 10μM for 1 h and Torin1 0.3 μM for 1 h as a positive control and re-fed with normal media plus imatinib and Torin1 for 2 h. n = 3 independent experiments. Scale bars, 10 μM. Statistical analysis with one-way ANOVA followed by Tukey's post-test. ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001. Data represent mean ± SEM.