Extended Data Fig. 2: Cnx and ERp57 are essential for ECM degradation.
a, Schematic of matrix degradation assay. b, Relative intensity of degraded collagen antibody staining after incubation with NIH 3T3vSrc control cells and NIH 3T3vSrc Cnx KD cells. Values indicate the mean ± SEM for 3 replicates and were analyzed using a two-tailed Student’s t-test. Representatives images can be found on the right. Scale bar, 20 μm. siRNA efficacy and its quantification can be found below. c, Workflow on ImageJ software to quantify degradation per nuclei. d, Quantification of ECM degradation assay of NIH3T3vSrc cells incubated with control antibody (IgG control) or 2 different anti-Cnx antibodies. Values indicate the mean ± SEM of normalized fold change for 3 replicates and were analyzed using a two-tailed Student’s t-test. e, Western blot analysis and quantification of cnx siRNA associated to Fig. 2d. Values were analyzed using a two-tailed Student’s t-test. f, Quantification of ECM degradation assay of NIH3T3vSrc cells transfected with control siRNA (siNT5) or siRNA against Cnx (siCnx). Values indicate the mean ± SEM of normalized fold changes for 3 replicates and were analyzed using a two-tailed Student’s t-test. Western blot and quantification of siRNA efficacy can be found on the right. g, Immunoblot analysis of MMPs protein level in NIH3T3vSrc transfected with control siRNA (siNT5) or siRNA against Cnx (siCnx). siRNA efficacy can be found on the right (this quantification is for Fig S2G, S2H and S2I, the experiments were done with the same samples). Values indicate the mean ± SEM of 3 replicates and were analyzed using a two-tailed Student’s t-test. h, Quantification of MMP activity by FRET assay of MDA ER-G2 cells transfected with control siRNA (siNT5) or siRNA against cnx (siCnx). Quantification of siRNA efficacy can be found on Supp Fig. 2g. i, Immunofluorescence of NIH3T3vSrc cells transfected with control siRNA (siNT5) or siRNA against Cnx (siCnx) and stained with MMP14 and the invadosome marker cortactin. Scale bar, 5 μm. Quantification of siRNA efficacy can be found on Supp Fig. 2g. j, Western blot and quantification of siRNA in Fig. 2h. Values indicate the mean ± SEM of 3 replicates and were analyzed using a two-tailed Student’s t-test. Source data and unmodified blots of Extended Data Fig. 2 can be found in files named Source Data Extended Data 2.
