Fig. 2: Role of Ca2+ and calcineurin in mitophagy.
a Tfeb-GFP-transfectants were transfected with pcDNA 3.1-HA or -HA-ΔCnA-H151Q. After rotenone or O/A treatment for 4 h, cells were subjected to immunofluorescence with anti-HA Ab. (scale bar, 20 μm) (white arrow, HA-ΔCnA-H151Q-transfected cells showing no TFEB translocation by rotenone or O/A; yellow arrow, untransfected cells showing TFEB translocation by rotenone or O/A; magenta arrow, control-transfected cells showing TFEB translocation by rotenone or O/A). b The percentage of cells showing nuclear TFEB in cells of (a). (scale bar, 20 μm) (n = 8 for HA + Rot or HA + O/A; n = 9 for HA + Veh; n = 19 for HA-∆CnA-H151Q + Rot; n = 20 for HA-∆CnA-H151Q + O/A; n = 21 for HA-∆CnA-H151Q + Veh) c mRFP-LC3-transfectants were transfected with pcDNA 3.1-HA or -HA-ΔCnA-H151Q. After rotenone or O/A treatment for 24 h, cells were subjected to immunofluorescence with anti-TOM20 and -HA Abs. (scale bar, 10 μm) d The number mRFP-LC3 puncta colocalized with TOM20 in cells of (c). (n = 7 for HA + Veh, HA + Rot, HA-∆CnA-H151Q + Rot or HA-∆CnA-H151Q + O/A; n = 8 for HA-∆CnA-H151Q + Veh; n = 9 for HA + O/A) e During GPN treatment of GCaMP3-ML1-transfected cells with or without rotenone or O/A pretreatment, perilysosomal fluorescence was traced (right upper). Peak fluorescence (right lower). Representative fluorescence images (left). (scale bar, 20 μm) (n = 5) f After mitochondrial stressor treatment for 4 h, [Ca2+]Lys was determined by Oregon Green 488 BAPTA-1 Dextran loading. Fluorescence intensity (right). Representative fluorescence images (left). (scale bar, 20 μm) (n = 7) g During GPN treatment of Fura-2-loaded cells with or without rotenone or O/A pretreatment, 340/380 nm fluorescence ratio was monitored (left). Lysosomal Ca2+ reservoir estimated by calculating AUC of the curve (right). (n = 14) h After treatment with mitochondrial stressors for 1 h, [Ca2+]i was determined using Fura-2. (n = 10) i After mitochondrial stressor treatment for 4 h in the presence or absence of BAPTA-AM, TFEB-GFP nuclear translocation was determined (right). Representative fluorescence images (left). (scale bar, 20 μm) (n = 5) 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 INS-1 cells or HA transfectants treated with Rot alone; ‡, compared to INS-1 cells or HA transfectants treated with O/A alone.
