Fig 2: No effect on p38a/ß MAPK by ZSWIM8 knockdown during C2C12 differentiation. (A) Control or 3 × FLAG-ZSWIM8-expressing C2C12 cells were differentiated for 1 or 2 days. The cell lysates were subjected to immunoprecipitation with an anti-FLAG antibody and immunoblotting with an anti-Cdon-N, Cdon-C, Bnip-2, JLP, FLAG, or Cul2 antibody. Cul2 and Ponceau S staining were used as loading controls. ZSWIM8-interacting Cdon fragments are indicated by arrows. Representative data of two independent experiments. (B) ZSWIM8 does not affect protein stabilities of Cdon, JLP, and Bnip-2. Control or ZSWIM8-knockdown C2C12 cells were differentiated for 3 days. The cells were further cultured with or without cycloheximide (CHX, 50 µg/mL) for the indicated time (h). The cell lysates were subjected to immunoblotting with an anti-Cdon-N, Cdon-C, JLP, Bnip-2, or ZSWIM8 antibody. Tubulin and Ponceau S staining were used as loading controls. Representative data of three independent experiments are shown. (C) Quantification of Cdon, Bnip-2, and JLP in (B). Cdon, Bnip-2, or JLP signals were normalized to that of Ponceau S staining. Expression in time = 0 was set as 1. Data represent the mean ± SD of three independent experiments. (D) Possible downregulation of p38a/ß MAPK phosphorylation by ZSWIM8 knockdown during C2C12 differentiation. Control or ZSWIM8-knockdown C2C12 cells were differentiated for 1 or 3 days. The cell lysates were subjected to immunoblotting with an anti-phospho-p38a/ß MAPK (pp38) or total p38a/ß MAPK antibody (p38). p38 and Ponceau S staining were used as loading controls. Representative data of five independent experiments are shown. (E) Quantification of p38a/ß MAPK phosphorylation in (D). pp38 signals were normalized to those of p38. The phosphorylation ratio in control cells without differentiation was set as 1. Data represent the mean ± SD of five independent experiments. Asterisk indicates statistical significance compared to the control sample. The membranes in (A), (B), and (D) were cut prior to hybridization with antibodies. Full-length blots are presented in Supplementary Fig. 11.

Fig 3: The proposed antifibrotic mechanism of BEL. BEL downregulates the phosphorylation of TßRI and TßRII, suppresses Smads2 and 4 expression and the phosphorylation and activation of Smads2 and 3 in CFs. Furthermore, BEL inhibits p38 phosphorylation and impedes NR4A1 cytoplasmic localization, which restores the inhibitory effect of NR4A1 to TGF-ß1-profibrotic signaling. These inhibitory effects further caused a decrease in the expression of a-SMA, collagen I, and collagen III, thereby preventing proliferation and activation of CFs to alleviate myocardial fibrosis.

Fig 4: The effects of BEL on p38 signaling in vitro and in vivo (A) The effects of different doses of BEL (15, 30, 60 µM) on the expression p38 and the level of P-p38 in CFs induced by TGF-ß1 using western blotting. Bar graphs show fold changes of P-p38/p38 (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, ## p < 0.01 vs. TGF-ß1 (B) The effects of BEL on the expression p38 and the level of P-p38 in vivo by western blotting. Bar graphs show fold changes of P-p38/p38 (n = 3). Data were shown as mean ± SEM. **p < 0.01 vs. control, ## p < 0.01 vs. ISO.