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. 2022 Feb 2;30(4):1432–1450. doi: 10.1016/j.ymthe.2022.01.037

Figure 4.

Figure 4

FLX activates TFEB in vitro

(A) Representative HCI images of HeLa stably expressing TFEB-GFP plasmid and untreated or treated with FLX for 3 h. (B) Representative image of immunoblot analysis of endogenous TFEB in MPS-IIIA MEFs untreated or treated with FLX, showing the shift from phosphorylated (pTFEB) to the dephosphorylated form of TFEB. As a positive control, cells are starved with HBSS. The plot shows the ratio between the 2 bands (TFEB/pTFEB). ANOVA test, ∗∗p < 0.01 versus untreated. (C) Graph of quantitative RT-PCR (qRT-PCR) showing the mRNA levels of a subset of TFEB target genes in MPS-IIIA MEFs upon 24 h of FLX treatment. The data in the graphs are mean values ±SEMs; n = 4 samples per condition. t test, ∗∗p < 0.01 versus untreated; ∗∗∗p < 0.001 versus untreated. (D) Representative HCI images of DQ-BSA assay in HeLa WT and TFEB/TFE3 KO untreated and treated with BafA1 or FLX. The plot shows the number of DQ-BSA spots per cell as means ± SEMs of n > 1,000 cells. ANOVA test, ∗∗∗p < 0.001 versus untreated. (E) Representative confocal images of HeLa stably expressing TFEB-GFP transfected with HA-GST-RagCS75L plasmid and treated with FLX for 3 h. The plot shows the nuclear:cytosol TFEB ratio as means ± SEMs of n > 50 cells from 3 independent experiments, in the 2 distinct populations, not transfected (HA−) and transfected (HA+, indicated with an asterisk). t test, ∗∗∗p < 0.001 versus HA−. (F) Representative HCI images of MPS-IIIA MEFs transfected with HA-GST-RagCS75L plasmid, pre-treated with FLX for 3 h and incubated overnight with DQ-BSA. The plot shows the DQ-BSA spots per cell as means ± SEMs of n > 1,000 cells from 3 independent experiments, in the 3 distinct populations, not transfected (HA−) and transfected (HA+, indicated with an asterisk). t test, ∗∗p < 0.01 versus HA−.