Fig 1: Versican-based growth differentiation factor 11 (GDF11) trapping is required for the proliferation-promoting effects of hyaluronan and proteoglycan link protein 1 (Hapln1). (A) Western blotting assays were conducted in the cytoplasmic and nucleus fractions to explore the effect of GDF11 manipulation on HAPLN1-induced SMAD2/3 phosphorylation in hiPSC-CMs (4 weeks, (4W)). Cells were treated with 100 ng/mL Hapln1 for 48 h, with or without GDF11 (20 ng/mL) or GDF11-neutralizing antibody (anti-GDF11, 100 ng/mL). (B, C) Western blotting assays were conducted to explore the expression of (B) the human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation markers (TNNI3 and MYL2), dedifferentiation markers (NKX2.5 and α-SMA) and (C) cell-cycle related proteins (P21, Cyclin D1, and Cyclin D2) in whole-cell lysate from hiPSC-CMs, as treated in Fig. 5A. (D) Western blotting assays were conducted of the cytoplasmic and nucleus fractions, to explore the effect of VCAN manipulation on HAPLN1-induced SMAD2/3 phosphorylation in hiPSC-CMs (4W). Cells with or without lentivirus-mediated VCAN knockdown were treated with 100 ng/mL HAPLN1 for 72 h. One VCAN knockdown group received both HAPLN1 (100 ng/mL) and GDF11 (20 ng/mL) treatment. (E, F) Western blotting assays were conducted to explore the expression of (E) the hiPSC-CM maturation markers (TNNI3 and MYL2), dedifferentiation markers (NKX2.5 and α-SMA) and (F) cell-cycle related proteins (P21, Cyclin D1, and Cyclin D2) in whole-cell lysate from hiPSC-CMs, as treated in Fig. 5D. (G–L) The proliferative abilities of hiPSC-CMs as treated in Figs. 5A and D, were investigated using EdU incorporation assays (G, I–J) and immunofluorescence staining of Ki67 (H, K–L). Two-way analysis of variance (ANOVA) with Bonferroni correction for post-hoc comparisons (G–J) was conducted. Data are presented as the mean ± standard deviation (n = 3). ∗P < 0.05. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; rhHapln1: recombinant human hyaluronan and proteoglycan link protein 1.