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. 2021 Mar 1;131(5):e143173. doi: 10.1172/JCI143173

Figure 5. Suppression of autophagy alleviates cardiac dysfunction in RagA/B cKO mice.

Figure 5

(A and B) CMs were transduced with Ad-lacZ, Ad-sh-RagA+Ad-sh-RagB, or Ad-sh-RagA+Ad-sh-RagB+Ad-sh-Atg7. (A) Cell viability quantified by CellTiter-Blue assay. n = 4. Values were measured from more than 8 different wells per experiment. (B) CMs were transduced with Ad-tandem fluorescent LC3, and accumulation of autophagosomes (yellow puncta) and autolysosomes (red puncta) was evaluated. n = 3. Values were measured for more than 50 cells per experiment. Scale bar: 20 μm. (C) Percentage of cells with PNS. n = 4–7. In each mouse, cells with PNS were counted from more than 100 CMs. (D) Cells were treated with DMOS, zVAD, or Nec1 with siRagA/B or siCont. Cell viability quantified by CellTiter-Blue assay. n = 4. In each experiment, cell viability was evaluated from more than 8 different wells. (E) HW/TL in WT, Atg7 hcKO, RagA/B cKO, and RagA/B cKO+Atg7 hcKO mice at 2.5 months old. n = 4–11. (F) Percentage of FS in WT, Atg7 hcKO, RagA/B cKO, and RagA/B cKO+Atg7 hcKO mice at 2.5 months old. n = 4–14. (G) WT and RagA/B cKO mice were injected with TAT-scrambled (TS) or TAT–beclin 1 (TB) for 1 to 2 weeks. Quantitative analyses of echocardiographically evaluated percentage of FS are shown. n = 4–12. (H) Neonatal CMs were treated with 5 μM TAT–beclin 1, 20 nM bafilomycin A1 or TAT–beclin 1+Bafilomycin A1 for 24 hours. Cells were stained with cardiac troponin T, and cell size was quantified. n = 6. In each experiment, cell size was measured from more than 50 cells. Results are expressed as mean ± SD. *P < 0.05; **P < 0.01, ANOVA.