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. 2021 Nov 10;12(11):1069. doi: 10.1038/s41419-021-04059-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A The subcellular distribution of SIRT1 (or Sirt1) was detected via subcellular fractionation and FISH assays. Scale bar, 10 μm. N = 3. B RT-qPCR analysis of circ-SIRT1 knockdown by sh-circ-SIRT#1/2 in hiPSC-CMs is shown. N = 3. C Analysis of the effects of circ-SIRT1 deficiency on cell surface area via immunofluorescence staining. Scale bar, 10 μm. N = 3. The percentage of cell surface area of the sh-circ-SIRT1#1 group to that of the sh-NC group was evaluated. D, E The expressions of CH markers in hiPSC-CMs transfected with sh-NC or sh-circ-SIRT1#1 were examined via RT-qPCR and western blot analyses. F Western blot analysis of levels of autophagy-related proteins (LC3-II/LC3-I, p62, and LAMP1) in hiPSC-CMs transfected with sh-NC or sh-circ-SIRT1#1. N = 3. G The hiPSC-CMs were transfected with Ad-GFP-mRFP-LC3 for 48 h and sh-NC or sh-circ-SIRT1#1. The change of both GFP fluorescence (green) and mRFP (red) fluorescence was observed by confocal microscopy. Scale bar, 10 μm. N = 3. H Circ-SIRT1 expression in hiPSC-CMs treated without or with Ang II and transfected with pcDNA3.1(+) or pcDNA3.1(+)/circ-SIRT1 (circ-SIRT1-oe) was examined via RT-qPCR. N = 3. I Cell surface area of hiPCS-CMs treated without or with Ang II and transfected with pcDNA3.1(+) or pcDNA3.1(+)/circ-SIRT1 (circ-SIRT1-oe) was analyzed by immunofluorescence staining. Scale bar, 10 μm. N = 3. J, K The expression of CH markers in hiPSC-CMs of the above-mentioned groups was examined via RT-qPCR and western blot analyses. N = 3. L, M The effect of upregulated circ-SIRT1 on autophagy influx in hiPSC-CMs treated without or with Ang II and transfected with pcDNA3.1(+) or pcDNA3.1(+)/circ-SIRT1 (circ-SIRT1-oe) was analyzed via western blot analysis of autophagy-related proteins and immunofluorescence staining analysis of Ad-GFP-mRFP-LC3. Representative images of GFP dots (green), mRFP dots (red), and their merged images are shown. Scale bar, 10 μm. N = 3. ^**P < 0.01 was assessed by Student’s t test for comparison between groups, one-way ANOVA and Tukey for multiple groups.