Figure 7. TFEB enters the nucleus in Protrudin- and FYCO1-depleted cells. (A) RPE-1 cells were grown in complete medium or starved in EBSS for 2 h, stained with an antibody to TFEB, and analyzed by high-content wide-field microscopy. (B) Automated quantification of images as in A of the mean intensity of nuclear TFEB using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 3). ***, P < 0.001 (unpaired t test). More than 1,700 cells were analyzed per condition. (C) RPE-1 cells grown in complete medium were transfected with control RNA or siRNA targeting Protrudin or FYCO1, stained with an antibody to TFEB, and analyzed by high-content wide-field microscopy. (D) Automated quantification of images as in C, of the mean intensity of nuclear TFEB using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05; **, P < 0.01 (unpaired t test). More than 1,200 cells were analyzed per condition. (E) HEK293 cells were transfected with control or Protrudin siRNA or treated with 500 nM Torin1 (1 h) or EBSS (2 h). Cytoplasmic and nuclear fractions were then prepared followed by immunoblotting analysis using indicated antibodies. ERK1/2 and histone H3 were used as the loading control for cytoplasmic and nuclear fractions, respectively. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 3). **, P < 0.01; ***, P = 0.001 (one-sample t test). (F) HEK293 cells were transfected with control or FYCO1 siRNA or treated with Torin1 (1 h) or EBSS (2 h). Separation of cytoplasmic and nuclear fractions and subsequent immunoblotting were performed as described in E. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05 (one-sample t test). (G) RPE-1 cells were treated as in E. Nuclear fractions were subjected to immunoblotting analysis with indicated antibodies. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 5). *, P < 0.05; **, P < 0.01 (one-sample t test). (H) RPE-1 cells were treated as in F. Nuclear fractions were analyzed using immunoblotting with the indicated antibodies. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-sample t test). (I) Protrudin depletion does not induce ER stress. RPE-1 cells were transfected with control RNA or siRNA targeting Protrudin and analyzed with immunoblotting using antibodies against the ER stress–induced proteins indicated. Thapsigargin was used to induce ER stress in nontransfected cells.
Figure 7.

TFEB enters the nucleus in Protrudin- and FYCO1-depleted cells. (A) RPE-1 cells were grown in complete medium or starved in EBSS for 2 h, stained with an antibody to TFEB, and analyzed by high-content wide-field microscopy. (B) Automated quantification of images as in A of the mean intensity of nuclear TFEB using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 3). ***, P < 0.001 (unpaired t test). More than 1,700 cells were analyzed per condition. (C) RPE-1 cells grown in complete medium were transfected with control RNA or siRNA targeting Protrudin or FYCO1, stained with an antibody to TFEB, and analyzed by high-content wide-field microscopy. (D) Automated quantification of images as in C, of the mean intensity of nuclear TFEB using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05; **, P < 0.01 (unpaired t test). More than 1,200 cells were analyzed per condition. (E) HEK293 cells were transfected with control or Protrudin siRNA or treated with 500 nM Torin1 (1 h) or EBSS (2 h). Cytoplasmic and nuclear fractions were then prepared followed by immunoblotting analysis using indicated antibodies. ERK1/2 and histone H3 were used as the loading control for cytoplasmic and nuclear fractions, respectively. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 3). **, P < 0.01; ***, P = 0.001 (one-sample t test). (F) HEK293 cells were transfected with control or FYCO1 siRNA or treated with Torin1 (1 h) or EBSS (2 h). Separation of cytoplasmic and nuclear fractions and subsequent immunoblotting were performed as described in E. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05 (one-sample t test). (G) RPE-1 cells were treated as in E. Nuclear fractions were subjected to immunoblotting analysis with indicated antibodies. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 5). *, P < 0.05; **, P < 0.01 (one-sample t test). (H) RPE-1 cells were treated as in F. Nuclear fractions were analyzed using immunoblotting with the indicated antibodies. Graphs represent quantifications of TFEB levels in nuclear fractions from immunoblots. TFEB levels were normalized to histone H3 and presented relative to control. Error bars denote ± SEM from independent experiments (n = 4). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-sample t test). (I) Protrudin depletion does not induce ER stress. RPE-1 cells were transfected with control RNA or siRNA targeting Protrudin and analyzed with immunoblotting using antibodies against the ER stress–induced proteins indicated. Thapsigargin was used to induce ER stress in nontransfected cells.

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