Fig. 5

Deprivation of SPARC in conditioned media from SPARC-expressing Ishikawa cells still activates normal fibroblasts. (A) Schematic presentation of the production of conditioned media from SPARC-expressing Ishikawa cells immunodepleted of SPARC using anti-FLAG antibody. (B) Successful immunodepletion of SPARC in conditioned media from control and SPARC-expressing Ishikawa cells was confirmed using immunoblotting. (C) Protein expression of ACTA2 and CDH2 in NF cultured in the conditioned media with or without immunodepletion of SPARC. Intensity of the bands was quantified using Image J. Values of the protein-of-interest were corrected using the intensity of GAPDH bands. NF were also analyzed for cell proliferation on day 6 (D) and in vitro contraction (E) in the conditioned media. (F–H) NF were treated with the indicated concentration of recombinant SPARC. Protein expression of ACTA2 and CDH2 (F), cell number on day 6 (G) and in vitro contraction ability (H) were assessed. NF, normal fibroblasts; Ctrl, control; rh SPARC, recombinant human SPARC; CM, conditioned media; n.s., not significant; *, P < 0.05; **, P < 0.01. Full-length blot images are presented in Supplementary Fig. 7, 8 and 9 (B, C, F). The experiments were independently repeated three times using identical fibroblasts and representative data were shown