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. 2022 Dec 20;5:1392. doi: 10.1038/s42003-022-04361-1

Fig. 4. ADAMTSL3 modulates cardiac TGFβ signalling in response to increased LV afterload in vivo and in human cardiac fibroblasts in vitro.

Fig. 4

a–c Wild-type (WT) and Adamtsl3 knock-out (L3-KO) mice were subjected to aortic banding (AB) or sham surgery for a total of 6 weeks. a,b Representative immunoblots and quantifications of active TGFβ1/total protein (a) and phosphorylated SMAD2 (pSMAD2)/total SMAD2 (b) in LV lysates, 1 week (sham: n = 4 WT and n = 4 KO, AB: n = 6-8 WT and n = 12 KO) and 6 weeks (sham: n = 4 WT and n = 4 KO, AB: n = 12 WT and n = 8 KO) post-AB. c mRNA/Rpl32 levels of TGFβ (Tgfb1), connective tissue growth factor (Ctgf), periostin (Postn) and latent TGFβ binding protein 1 (Ltbp1) in AB mice vs sham 6 weeks post-AB (Sham: n = 8 WT and n = 8 KO, AB: n = 10 WT and n = 7 KO). Statistical analyses waere performed using the one-way ANOVA with Tukey’s multiple comparisons test, with *p < 0.05, **p < 0.01, and ***p < 0.001 for AB vs. sham, and $p < 0.05, $$p < 0.01, and $$$p < 0.001 for L3-KO AB vs. WT AB. d-k Human foetal cardiac fibroblasts cultured for 7 days, producing a rich extracellular matrix (ECM) network, and transduced with ADAMTSL3 (L3) or control (vehicle, Veh) adenovirus on day 4. Data represent experiments from 3 different cell passages. d Representative immunoblot and quantification of ADAMTSL3/total protein in Veh (n = 9) and L3 (n = 11) lysates. e 62 downregulated and 22 upregulated genes in pooled L3 vs Veh mRNA samples (n = 18 in both groups) using an expression array of 84 TGFβ related genes, data are ΔΔCT values normalized to GAPDH. f–g mRNA/RPL4 levels from qPCR of TGFB1, LTBP1, CTGF, POSTN (f) and MALAT1 (g) in L3 (n = 16) vs Veh (n = 18). h–k Representative immunoblot and quantification of pSMAD2/SMAD2 (h), active TGFβ1/total protein (i), latency associated protein (LAP)/total protein, including the large latent complex (LLC, > 250 kDa) (j), and latent TGFβ binding protein (LTBP1)/GAPDH, including the LLC (k), in L3 (n = 8-9) vs. Veh (n = 8) cell- and extracellular matrix lysates. Statistical analyses were performed using the Student’s t-test (d, f-k). All data (except e) are shown as individual value scatterplots with mean ± SEM.