Fig. 2: MFAP5 knockdown inhibits CAFs activation and tumor progression in vitro.

A Relative expression of CAF activation-related genes detected with qRT‐PCR. B, C Immunofluorescence staining of MFAP5 and α-SMA in human MFAP5_KD or control CAFs with or without TGF-β (5 ng/ml for 24 h). Scale bar: 100 μm (D) Western blotting assay of CAF activation in MFAP5_KD or control ImdyCAFs treated with TNF-α or IL-6 (10 ng/ml for 48 h). Arrow indicates the true protein band. E Morphological characteristics and changes in diameter measured in day 2 and day 5 in spheroid formation assay. Statistical of diameter at day 2 is shown on the right. Scale bar: 50 μm. F Trans-well experiment indicates migration and invasive capabilities of Panc-1 and Panc02 in the conditioned medium. Panc-1 was co-cultured with CAF3 control or KD lines, and Panc02 was cultured with ImdyCAF control or KD lines. Scale bar: 50 μm. G Western blot assay of EMT-related proteins in Panc-1 and Panc02 from the cocultured systems. H Representative images of 2D/3D tube formation assays of HUVEC cells in cocultured system and statistical results. Scale bar: 50 μm. I–N Representative images of heterospheroids derived from Panc02 or KPC and stable transfected ImdyCAFs after treatment of gemcitabine (10 μM) (I and L), and the statistical results of spheroid volume (J and M) and cell viability (K and N). O Western blotting assay of gemcitabine transport proteins in Pand02 and KPC after cocultured with conditioned medium for 48 h. Gray values of the protein bands were calculated with Image J and the relative expression level were labeled. The data were analyzed by a two‐tailed unpaired Student’s t‐test (A, B, E, F, G, H, J, K, M and N). Error bars, means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant.