Fig 2: AcSDKP suppresses TGFβ/smad signaling and EndMT through the FGFR1/FRS2 pathway. (a) HMVECs were treated with N-FGFR1 for 48 h, and the FGFR1, TGFβR1 and TGFβR2 protein levels were analyzed by western blot. (b) HMVECs were treated with TGFβ2 in the presence or absence of N-FGFR1 for 15 min with or without AcSDKP preincubation. The p-smad3 and TGFβR1 protein levels were analyzed by western blot. Densitometric analysis of the p-smad3/smad3 and TGFβR1/β-actin levels (n=3) in each group was performed. (c) HMVECs were incubated with either N-FGFR1 in the presence or absence of TGFβ2 for 48 h with or without preincubation with AcSDKP for 2 h or with N-FGFR1 in the presence or absence of TGFβ2 for 48 h with or without 24 h of incubation with FGF2 (50 ng/ml). The CD31, SM22α, FSP1 and α-SMA protein levels were analyzed by western blot. (d) HMVECs were transfected with FRS2 siRNA (100 nM) for 48 h with or without AcSDKP preincubation. The VE-cadherin, FSP1, vimentin, SM22α and p-smad3 levels were analyzed by western blot. (e) HMVECs were treated with N-FGFR1 for 48 h or 15 min in the presence or absence of N-TGFβ (1, 2, 3) (1.0 μg/ml). The CD31, VE-cadherin, SM22α, FSP1, TGFβR1, TGFβR2 and p-smad3 levels were analyzed by western blot

Fig 3: MAP4K4 deficiency induces TGFβ/smad signaling and EndMT via activation of integrin β1. (a) HMVECs were transfected with MAP4K4 siRNA (100 nM) for 48 h. Next, the cells were treated with or without AcSDKP for 2 h. The p-smad3/smad3 pathway was analyzed by western blot. Densitometric analysis of the p-smad3/smad3 levels was performed, with n=3 for each group. (b) HMVECs were treated with MAP4K4 siRNA for 48 h with or without AcSDKP treatment. The VE-cadherin, CD31, FSP1, SM22α and vimentin protein levels were analyzed by western blot. (c) HMVECs were transfected with MAP4K4 siRNA for 48 h in the presence or absence of TGFβ2 with or without AcSDKP. The integrin β1 level was analyzed by western blot

Fig 4: Treatment with a Smad3 inhibitor SIS3 inhibits SARS‐CoV‐2 N protein‐induced AKI in mice. A) PAS staining and semiquantitative analysis of tubular necrosis. B) Serum creatinine. C) Western blotting for activation of Smad3 (p‐Smad3) and expression of p21 and cyclin E protein. D) Real‐time PCR for mRNA levels of SARS‐CoV‐2 N, p21, and cyclin E expression. E) Immunohistochemistry for detecting SARS‐CoV‐2 N protein expression, activation of Smad3 signaling (p‐Smad3 nuclear translocation), and cell apoptosis by TUNEL‐labeling. Each dot represents one mouse and data are expressed as the mean ± SEM for groups of six mice. *p < 0.05, ***p < 0.001 versus control mice; ##p < 0.01, ###p < 0.001 versus SARS‐CoV‐2 N plasmid with DMSO (N+DMSO) group. g, glomerulus; scale bars = 50 µm .

Fig 5: Smad3 deficiency suppresses cardiac inflammation in db/db mice. A, Representative immunohistochemical images of F4/80, IL‐1β, TNF‐α and MCP‐1. B‐E, Quantitative analysis of immunohistochemical staining of F4/80, IL‐1β and TNFα. F‐H, Real‐time PCR analysis of IL‐1β, TNF‐α and MCP‐1 mRNA expression. Values are expressed as mean ± SE for group of eight mice. *P <.05, **P 01 and ***P 001 compared with Smad3 (S3) WT‐db/m group; # P 05, ## P 01 and ### P 001 compared with Smad3 (S3) WT‐db/db and Smad3+/−db/db groups. Scale bar = 20 µm