Figure 4.

PROX1 silencing increases MMP14 expression and MMP14-dependent 3D sprouting in cancer cells. (a) Chromatin immunoprecipitation in HepG2 and SW620 cells ectopically expressing Myg-tagged PROX1. Chromatin was immunoprecipitated using either an anti-Myc antibody or an irrelevant anti-HA antibody and subsequently amplified by qPCR for the indicated regions of the MMP14 promoter (illustrated in Fig. 2b). Average fold enrichment over the anti-HA control is shown for three independent experiments; error bars represent SD. (b) HepG2 and SW620 cells were transfected with the indicated siRNAs for 48 h and whole cell extracts were analysed by RTqPCR for the indicated targets with GAPDH as an internal control. Bars represent an average of two independent experiments, error bars show SD across the experiments. (c) HepG2 and SW620 cells were treated as in (b) and whole cell extract was analysed by immunoblot for the indicated proteins using actin as a loading control; numbers indicate the intensity of the MMP14 band normalized to the corresponding loading control. Cropped membranes are shown, uncropped membranes can be found in Fig. S3c,d. (d,e) 3D fibrin invasion assay with HepG2 cells transfected twice in consecutive days with the indicated siRNAs and embedded in 3D fibrin in the presence of either DMSO or 50 μM of the MMP14 inhibitor NSC405020. After 4 days, fibrin gels were fixed and stained with Phalloidin (Phall A488) and Hoechst 33342. (d) Representative images are shown. (e) Quantification of three images per condition from two independent experiments (n > 100 cells/condition). Bars represent the average area occupied by each cell cluster and normalized to the scr siRNA and DMSO-treated sample. n.s.: non-significant; **p > 0.01; ***p > 0.001.