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. Author manuscript; available in PMC: 2021 Oct 21.
Published in final edited form as: Sci Transl Med. 2020 Oct 21;12(566):eaay7856. doi: 10.1126/scitranslmed.aay7856

Fig. 5. Becn1 inhibits iCM conversion independent of autophagy.

Fig. 5.

(A) Schematic of experimental design to determine the temporal requirement of Becn1 knockdown in cardiac reprogramming. (B) Quantification of αMHC-GFP% and cTnT% on reprogramming day 10 as designed in (A). (C) qPCR of gene expression at different time points upon MGT transduction and Becn1 knockdown. Values were normalized to uninfected control on day 3 (mock). (D) Western blot analysis of αMHC-GFP, Mef2c, Gata4, and Tbx5 expression. β-Actin serves as a loading control. (E) Schematic of experiment design to determine the time window of Becn1 knockdown. (F) Fold change (FC) of αMHC-GFP% and cTnT% in iCMs as described in (E). (G and H) Representative images showing GFP-LC3 puncta formation (G) and quantification (H) in iCMs transduced with shNT or shBecn1 on reprogramming day 5. Lentiviral GFP-LC3 constructs were transduced into iCMs on reprogramming day 1. (I and J) Representative Western blot (I) and quantification (J) showing the autophagy flux in iCMs treated with shNT or shBecn1 from reprogramming day 3 to day 14. Sample on the indicated dates were treated with or without CQ for 4 hours. β-Actin serves as loading control. wo/CQ, without CQ treatment; w/CQ, with CQ treatment. (K) Representative images showing autophagosome (yellow) and autolysosome (red) in iCMs transduced with mRFP-GFP-LC3 construct. shRNAs targeting Becn1 or control shRNA (shNT) were introduced into iCMs on day 1, and images were taken on reprogramming day 5 after cells were treated with or without CQ for 4 hours. (L) Quantification of autophagosome ratio in (K). (M and N) FACS analysis (M) and quantification (N) of iCMs upon lentiviral delivery of shNT, shBecn1, shAtg5, and shBecn1 plus shAtg5 on reprogramming day 10. All experiments were repeated at least three times. Mean values from technical triplicates [except n = 10 to 15 for (H) and (L)] were used for statistics. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test for multiple groups. Error bars indicate means ± SEM; **P < 0.01 and ***P < 0.001.