Fig. 4

Arf6-mediated CD147 recycling facilitates cell adhesion, aggregation, and tight junction formation. Arf6-perturbed 7721 cells were detached and reseeded on Matrigel- (a), collagen- (b), fibronectin- (c) or laminin- (d) coated plates, and the attached cell number was determined. Three independent experiments were performed in quintuplicate. Significant differences compared with NC-KD cells are shown. OD: optical density. Arf6-perturbed 7721 cells were reseeded on agar substrate for 24 h static culture. Cell aggregation clusters were evaluated microscopically. e Representative light microscopy pictures are shown (×40). f The number of different-sized clusters was compared among groups. Sixty fields from three independent experiments were counted and analyzed. Significant differences compared with NC-KD cells are shown. Arf6-perturbed 7721 cells were confluent grown. Expressions of tight junction marker (ZO-1) and adherent junction marker (E-cadherin) were determined. Representative blot results from three independent experiments are shown (g). Protein bands were quantified, and the expression level in untreated cells (WT) was set as 100%. Significant differences compared with NC-KD cells are shown (h). * P < 0.05, ** P < 0.01. In parallel, the treated cells were respectively stained with H18Ab (coupled with anti-mouse AF488), ZO-1 Ab (coupled with anti-rabbit AF647), and E-cadherin Ab (coupled with anti-mouse AF647), and viewed by confocal microscopy. Optical sections at the plane of cell junctions are shown (i). Vertical (x/y) fields of CD147-stained Huh7 cells along the z-axis are also shown (j). Supernatants of cultured cells were concentrated (20 fold) and loaded for CD147 blotting. Representative results from three independent experiments are shown (k), and corresponding quantitative data were analyzed (l)