Extended Data Fig. 5: Citrulline inhibits PAD4-mediated TGF-β1 citrullination.
a, Western blot showing the citrullination level of TGF-β1 in MPFs treated with PBS or indicated EVs, total TGF-β1 level served as a loading control. b, Real-time qPCR analysis of Padi2 mRNA abundance in WI-38/PADI2 KD cells compared with WI-38 cells transfected with plko.1 vector; n = 3 biologically independent samples per group. Values were normalized to vector control. Data were presented as mean ± s.e.m. c, Real-time qPCR analysis of Padi4 mRNA abundance in WI-38/PADI4 KD cells compared with WI-38 cells transfected with plko.1 vector; n = 3 biologically independent samples per group. Values were normalized to vector control. Data were presented as mean ± s.e.m. d-e, Western blot showing indicated protein levels in WI-38/PADI2 KD cells and WI-38/PADI4 KD cells following 48-hour exposure to 20 μM citrulline or PBS (solvent control) compared with WI-38 cells transfected with plko.1 vector; β-Actin served as a loading control. f-g, Western blot showing the citrullination level of TGF-β1 in WI-38 cells with PAD2 or PAD4 knockdown (WI-38/PADI2 KD or WI-38/PADI4 KD) following 24-hour exposure to 20 μM citrulline or PBS (solvent control) compared to plko.1 vector-transfected controls. Total TGF-β1 level was used as a loading control. h, Genotype identification of Padi4−/− mice. i, MPFs treated with PBS, indicated EVs or 20 μM citrulline were subjected to SMAD3 immunofluorescence staining and confocal microscopic imaging. Representative images were shown. Scale bar, 10 μm. j, MPFs from wild-type C57BL/6 J mice or Padi4−/− mice were treated with 20 μM citrulline or PBS to detect the amounts of t-SMAD3 (total SMAD3), p-SMAD3 (phosphorylated of SMAD3 at S423/S425) in the nucleus and cytoplasmic fractions by western blot. H3 (a nuclear control) or β-Actin (a cytoplasmic control) served as a loading control. Blots were quantified with ImageJ (quantification of proteins in nucleus or cytoplasmic fractions normalized to respective loading control and shown below image). k, Western blot showing the citrullination level of TGF-β1 in wild-type and Padi4−/− MPFs following 24-hour exposure to 20 μM citrulline or PBS (solvent control), total TGF-β1 level served as a loading control. l, Western blot showing the citrullination level of H2A, H3, H4, and GSK3β in wild-type and Padi4−/− MPFs exposure to 20 μM citrulline or PBS (solvent control), total indicated protein level served as a loading control. Blots were quantified with ImageJ (values under each blot were normalized to the corresponding control and subsequent to the first lane). m, Population doubling curves of indicated MPFs isolated from Padi4−/− mice; n = 3 biologically independent samples per group. n, Representative H&E staining images and corresponding quantitative analysis of lung tissues in Padi4−/− C57 mice from PBS/citrulline intranasal administration model (Scale bar, 200 μm; n = 8 mice per group). Data were presented as mean ± s.e.m. o, Representative Sirius red staining images and corresponding quantitative analysis of lung tissues from Padi4−/− C57 mice with PBS/citrulline intranasal administration (Scale bar, 100 μm; n = 7 mice per group). Data were presented as mean ± s.e.m. p, Representative IHC staining images and corresponding quantitative analysis of DDAH1 in indicated EV injection group Padi4−/− mice lung tissues (Scale bar, 100 μm; n = 7 mice per group). Data were presented as mean ± s.e.m. q, Representative images and corresponding quantitative analysis of H&E staining (Scale bar, 100 μm; n = 7 mice per group). Data were presented as mean ± s.e.m. r, Representative Sirius red staining images and corresponding quantitative analysis of lung tissue in indicated EV injection group Padi4−/− mice (Scale bar, 200 μm; n = 7 mice per group). Data were presented as mean ± s.e.m. s, Western blot showing the citrullination level of TGF-β1 in WI-38 cells transfected with wild-type PAD4 or the E411A and R651A double mutated PAD4 following 24-hour exposure to 20 μM citrulline or PBS, total TGF-β1 level served as a loading control. Experiments were repeated three times for a, d-g, j-l, s. Representative images are displayed. Data were presented as mean ± s.e.m.; exact P values were shown and reported as source data. One-way ANOVA followed by Dunnett multiple comparison test was used for b, c, p, q, r; two-way ANOVA was used for m; unpaired two-tailed Student’s t-test was used for n, o.
