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. 2021 Oct 29;12:741219. doi: 10.3389/fphar.2021.741219

FIGURE 6.

FIGURE 6

SC75741 activates TFEB independently of mTORC1 activity. (A). H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, imatinib (5 μM) or SC75741 (5 μM) for another 24 h. Nuclear lysates and cytoplasmic lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (TFEB/Tubulin, TFEB/H3). (B). H4GT25 cells were pretreated with 1 µg/mL DOX for 24h, treated with SC75741 (5 μM) for 12 and 24 h. Nuclear lysates and cytoplasmic lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (TFEB/Tubulin, TFEB/H3). (C). H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, imatinib (5 μM) or SC75741 (5 μM) for another 12h, and then the cells were subjected to immuno-fluorescence analysis using an anti-TFEB antibody. Scale bar, 40 μm. (D). Quantification of the percentage of TFEB-positive cells in the nucleus and TFEB-positive cells in the cytoplasm. (data represents mean ± SD; DMSO (n =110 cells), imatinib (n =112 cells), SC75741 (n =101 cells)). (E). H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, Torin1 (1 μM), imatinib (5 μM) or SC75741 (5 μM) for another 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (TFEB/Tubulin). (F). q-PCR analysis of different mRNA TFEB target genes in H4GT25 cells treated with imatinib (5 μM) and SC75741 (5 μM) for 12 h. (G). H4GT25 cells were pretreated with 1 μg/ml DOX for 24 h, treated with DMSO, Torin1 (1 μM), imatinib (5 μM) or SC75741 (5 μM) for another 12 h. Cell lysates were immunoblotted with indicated antibodies. The numbers under the blot represent the gray scale quantification (p-S6K/Tubulin, S6K/Tubulin,).