Fig. 4

The ANXA2/CTGF-TSP1 complex is required on cell proliferation, migration, and angiogenesis in vivo and in vitro. A, B The expression of CTGF in wild-type FLS and CTGF-KD FLS were measured by WB. The experiments were repeatedly performed using three independent samples. C The expression of Flag-CTGF/CTGF-∆TSP1 and ANXA2 in CTGF-KD FLS transfected with lentivirus (LV-CON, LV-Flag-CTGF-∆TSP1, LV-Flag-CTGF-full length, LV-sh-ANXA2+Flag-CTGF-full length, LV-sh-ANXA2+Flag-LV-CTGF-∆TSP1) were measured by WB. D MTT assay. CTGF-KD FLS were transfected with lentivirus (groups mentioned above in Fig. 4C, n = 3) and then incubated for 6 h, 12 h, 18 h, and 24 h. The absorbance value of each well at the 490-nm wavelength was measured. E Scratch migration assay. CTGF-KD FLS were seeded on a six-well plate and transfected with lentivirus (groups mentioned above in C, n = 3). After 24 h, a scratch was made along the diameter of the well using a pipette tip. An image was captured every 12 h by a camera under an inverted microscope (original magnification × 40). F The percentage of wound closure at 24 h was quantified as (area of original wound − area of measured wound)/area of original wound × 100%. G Tube formation assay. HUVECs were transfected with lentivirus (LV-Flag-CTGF-full length, LV-Flag-CTGF-∆TSP1, and LV-sh-ANXA2+Flag-CTGF-full length) and then incubated for 6 h, and then tube formation was observed and photographed (n = 10). H The number of intersections among the branches of assembled HUVEC networks was calculated in the whole field. I CAM assay. CAMs were treated with lentivirus (groups mentioned above in G) on day 7, and all CAMs were photographed 3 days later (n = 10). J The percentage of angiogenetic area = vascular area/CAM area × 100%. ImageJ was used to assess the vascular and CAM areas