Fig 2: cANGPTL4 upregulates ID3 via a ß-catenin-dependent pathway. (a) Immunoblot of full-length cadherin-11 (CDH11) and splice variant CDH11 (CDH11var) in human fibroblasts. Fibroblasts were transfected with empty vector (Fctrl) or expression vector containing cDNA encoding CDH11var (FCDH11var). ß-tubulin, as loading control, was from the same samples. Relative mRNA expression of ID3 in fibroblasts subjected to the indicated treatments. (b–e) Immunodetection and quantification of ID3 or ß-catenin proteins in fibroblasts subjected to the indicated treatments. ß-tubulin (cytosol) and c-JUN (nuclear) from the same samples were used as loading controls. Immunoblot images are cropped; full-length immunoblots can be found in Supplementary Fig. S4h–n. (f,g) Relative mRNA and protein expression of ID3 in fibroblasts treated with ICG001 and XAV939, two inhibitors of ß-catenin downstream signaling. Ribosomal protein L27 was used as housekeeping control for qPCR. ß-tubulin from the same samples was used as loading control for immunoblots. Values represent mean ± SD, n = 5 independent experiments. n.s., not significant, *P < 0.05, **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). (h) ChIP of TBE on the human ID3 gene using anti-ß-catenin antibody followed by re-ChIP with anti-LEF antibody. Gene sequence spanning the TBE II/III and a random control sequence were analyzed by PCR in the immunoprecipitated chromatin of fibroblasts subjected to the indicated treatments. Pre-immune serum served as control. PCR using chromatin before immunoprecipitation served as input. M, 100 bp DNA ladder. (i,j) Immunodetection of ID3, ScxA and E47 in the lysates and immunoprecipitates of day 5 and 10 cANGPTL4-treated wounds (n = 9). Values were normalized against total protein or ID3 anti-IgG antibodies. Loading controls were from the same samples/immunoprecipitates. Representative immunoblot images are cropped; full-length immunoblots scanned using the Li-Cor Odyssey® CLx system can be found in Supplementary Fig. S5. (k) Illustration depicting the mechanistic action of cANGPTL4 on COL1A2 expression in fibroblasts. cANGPLT4 interacts with fibroblast CDH11, triggering ß-catenin nuclear translocation, where it interacts with TCF/LEF to transcriptionally activate ID3 gene expression. ID3 heterodimerizes with scleraxis to inhibit the transcription of COL1A2, reducing collagen secretion.