Fig. 1: Mitophagy after treating INS-1 cells with mitochondrial stressors. | Nature Communications

Fig. 1: Mitophagy after treating INS-1 cells with mitochondrial stressors.

From: Lysosomal Ca2+-mediated TFEB activation modulates mitophagy and functional adaptation of pancreatic β-cells to metabolic stress

Fig. 1

a INS-1 cells transfected with pMito-Keima were treated with rotenone or O/A for 18 h. Keima fluorescence at neutral pH (green) and in acidic conditions (red) was determined, and the number of red puncta indicating mitophagy was counted (right). Representative fluorescence images are presented (left). (scale bar, 5 μm) (n = 9) (Veh, vehicle; Rot, rotenone; O/A, oligomycin + antimycin A) b INS-1 cells transfected with mRFP-LC3 were treated with rotenone or O/A for 24 h and then subjected to immunofluorescence using anti-TOM20 Ab to visualize the colocalization of an autophagic marker with a mitochondrial protein, i.e., mitophagy (right). Representative fluorescence images are presented (left). (scale bar, 5 μm) (n = 5) c INS-1 cells transfected with TFEB-GFP or TFE3-GFP were treated with rotenone or O/A for 4 h, and then cells with nuclear translocation of TFEB-GFP or TFE3-GFP were counted by confocal microscopy (right). Representative fluorescence images are presented (left). (scale bar, 20 μm) (n = 5 for TFEB-GFP; n = 3 for TFE3-GFP) d INS-1 cells were treated with rotenone or O/A for 4 h, and then cells with total or partial nuclear translocation of endogenous TFEB or TFE3 were counted after immunostaining with anti-TFEB or -TFE3 Ab (right). Representative fluorescence images are presented (left). (scale bar, 20 μm) (n = 4 for TFEB; n = 5 for TFE3) e Tfeb-KO and Tfe3-KO INS-1 cells generated by CRISPR/Cas9 technology were transfected with pMito-Keima, and then treated with rotenone or O/A for 18 h. Keima fluorescence at neutral pH and in acidic condition was determined by confocal microscopy (left). The number of red puncta indicating mitophagy was counted (right). (scale bar, 5 μm) (n = 7 for Tfeb KO + O/A; n = 9 for Tfeb KO; n = 10 for Tfeb KO + Rot; n = 15 for O/A or Tfe3 KO; n = 16 for Tfe3 KO + O/A; n = 18 for Tfe3 KO + Rot; n = 20 for Con or Rot) (Con, Cas9 control) Cells in the rectangles were magnified. All data in this figure are the means ± SEM from more than 3 independent experiments. P values were determined using one-way ANOVA with Tukey’s test. *, compared to Veh-treated cells; †, compared to Rot-treated Con cells; ‡, compared to O/A-treated Con cells.

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