Fig. 1: Activation of TFEB/TFE3 by MSL-7.

a INS-1 cells were transfected with mRFP-GFP-LC3. The numbers of yellow (autophagosomes) and red puncta (autophagolysosomes) were counted (t = 9.8, df = 38 for autophagosome; t = 17.2, df = 38 for autophagolysosome) (right). Representative pictures are presented (left). Inset images were magnified. b INS-1 cells were treated with MSL-7 in the presence or absence of bafilomycin A1 (BAF). Immunoblotting using the indicated antibodies (ACTB, β-actin) were conducted. Numbers indicate fold changes normalized to ACTB bands. c–d Nuclear translocation of TFEB (c) and TFE3 (d) by MSL-7 was counted (t = 29.9, df = 12 in c; t = 38.7, df = 12 in d) (right of c, d). Representative pictures are shown (left of c, d). e INS-1 cells were treated with MSL-7, and immunoblotting using the indicated antibodies were conducted. Numbers indicate fold changes normalized to total TFEB bands. f After lysis of INS-1 cells treated with MSL-7 for 4 h, immunoprecipitation (IP) was conducted using anti-TFEB (left) or anti-TFE3 antibody (right). Supernatant was subjected to immunoblot analysis (IB) using the indicated antibodies. Numbers indicate fold changes normalized to total TFEB or TFE3 bands. g Nuclear and cytosolic fractions of lysates from INS-1 cells treated with MSL-7 were subjected to immunoblot analysis using the indicated antibodies. Numbers indicate fold changes normalized to ACTB bands (cytosol) or Lamin bands (nuclear). h After lysis of Tfeb-GFP-transfected (left) or Tfe3-GFP-transfected cells (right) treated with MSL-7, IP using anti-GFP antibody was conducted. i After treatment of Tfeb-GFP-transfected or Tfe3-GFP-transfected cells with MSL-7, confocal microscopy was conducted. Numbers indicate fold changes normalized to GFP bands. j Real-time RT-PCR was performed using mRNA from INS-1 cells treated with MSL-7 for 6 h and specific primers. All data in this figure are the means ± SEM from more than 3 independent experiments performed in triplicate. (scale bar, 5 μm) *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student’s t-test (a, c, d, j). Source data are provided as a Source Data file.