Fig 1: MasR activation by CAP-1902 triggers the AMPK/ULK1/FUNDC1 axis to increase mitophagy.(A) Representative blots of phosphorylated and total AMPK in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 10 and 30 minutes. Vinculin was used as loading control. (B) Average expression of p-AMPK by total AMPK levels in Ctrl vs CIII-deficient, and (C) CIII-deficient untreated and treated with CAP-1902. Statistical analysis was performed using a t-test and one-way ANOVA, respectively. (D) Representative blots of phosphorylated and total AMPK in CIII-deficient untreated and treated with 1 μM A779 for 1h, plus and minus 5 nM CAP-1902 for the last 30 minutes. (E) Average expression of p-AMPK by total AMPK levels. Statistical analysis was performed by one-way ANOVA. (F) Representative blots of phosphorylated (S638) ULK1 and total ULK1 in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. ACTIN was used as loading control. (G) Average expression of p-ULK1 by total ULK1 levels in Ctrl vs CIII-deficient untreated and (H) CIII-deficient untreated and treated with CAP-1902. Statistical analysis was performed by unpaired t-test. (I) Representative blots of ULK1 in mitochondria-enriched fractions (M) vs the corresponding supernatant (S) in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. Citrate synthase is included as a control for mitochondrial enrichment, and total protein stain as loading control. (J) Quantification of the expression of ULK1/total protein in each fraction. Statistical analysis was performed by two-way ANOVA. (K) Representative blots of phosphorylated (S17) FUNDC1 and total FUNDC1 in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. ACTIN was used as loading control. (L) Average expression of p-FUNDC1 by total FUNDC1 levels in Ctrl vs CIII-deficient untreated and (M) CIII-deficient untreated and treated with CAP-1902. Statistical analysis was performed by unpaired t-test. (N) Number of mitolysosomes in Ctrl, CIII-deficient, and CIII-deficient cells treated with 5 nM CAP-1902 for 16 h. Quantification represents the average number of LC3-II puncta touching mitochondria (mitophagosomes) per cell. Statistical analysis was performed by two-way ANOVA. (O) Confocal micrographs of Ctrl, CIII, and CIII cells treated with 5 nM CAP-1902 ± bafilomycin for 16 h, labeled with LC3B (autophagosome – red), GRP75 (mitochondria - green), and DAPI (nuclei - blue). Scale bars: 100 μm and 5 μm in the zoomed image. In all cases, data show mean ± SEM. Dots in graphs represent independent biological replicates.
Fig 2: FUNDC1 knockdown abolishes MasR induced mitophagy.(A) Representative high-throughput confocal micrographs of CIII-deficient cells with shRNA knockdown for FUNDC1 (CIIIFUNDC1_KD) untreated and treated with 5 nM CAP-1902 for 16h. Autophagy flux is blocked using 100 nM bafilomycin for 16 h. Scale bars: 100 μm and 5 μm in the zoomed image. (B) Number of mitolysosomes in CIII-deficient and CIIIFUNDC1_KD. Quantification represents the average number of LC3-II puncta touching mitochondria (mitophagosomes) per cell. Statistical analysis was performed by two-way ANOVA. (C) Number of mitophagosomes in CIIIFUNDC1_KD untreated and treated with 5 nM CAP-1902 for 16h. Quantification represents the average of LC3-II puncta at mitochondria per cell. Statistical analysis was performed by two-way ANOVA. (D) Representative blots of phosphorylated and total AMPK in CIIIFUNDC1_KD untreated and treated with 5 nM CAP-1902 for 10 and 30 minutes. ACTIN was used as loading control. (E) Average expression of p-AMPK referred to total AMPK in the conditions specified in D. (F) Representative confocal micrographs of CIIIFUNDC1_KD untreated and treated with 5nM CAP-1902 for 16 h labeled with PGC1α antibody (red) and DAPI (nuclei – blue) (top). Mask showing the intensity of the nuclei in the previous images (bottom). Scale bars: 100 μm. (G) Quantification of the average PGC1α fluorescence intensity in the nuclei. Statistical analysis was performed by paired t-test. (H) Representative confocal micrographs of CIII-deficient, CIIIFUNDC1_KD cells untreated and treated with 5 nM CAP-1902 for 16 h labeled with GRP75 antibody (mitochondria – red) and DAPI (nuclei – blue) (top). GFP signal from the same samples indicates infection with FUNDC1 shRNA (bottom). Scale bars: 100 μm. (I) Quantification of mitochondrial mass in CIIIFUNDC1_KD untreated and treated with 5 nM CAP-1902 for 16 h compared to CIII-deficient cells (100%). Statistical analysis was performed by paired t-test. In all cases, data show mean ± SEM. Dots in graphs represent independent biological replicates.
Supplier Page from OriGene Technologies for FUNDC1 Human shRNA Lentiviral Particle (Locus ID 139341)