Fig. 1

TSP-4 is mainly derived from CAFs in GBC and predicts poor prognosis. a Immunofluorescence (IF) staining of α-SMA in human GBC and adjacent non-tumor (NT) tissue. The magnification of the picture is 400×. Scale bars = 20 μm. b CAFs and PTFs were isolated from human GBC tissues and adjacent non-tumor tissue. α-SMA level in CAFs and PTFs were assessed by IF staining. The magnification of the picture is 400×. Scale bars = 20 μm. c, d The expression of α-SMA, fibronectin and col. 1α at mRNA and protein level was determined by qRT-PCR and Western blot respectively. n = 15, *P < 0.05 or **P < 0.01 by Student’s t-test. e qRT-PCR was used to screen the expression of thrombospondins (TSP1-TSP5) in CAFs and PTFs. n = 15, *P < 0.05 or **P < 0.01 or ***P < 0.001 by Student’s t-test. f TSP-4 level in CAFs and PTFs were determined by IF staining. The magnification is 400×. Scale bars = 20 μm. g The secretion of TSP-4 in GBC cell lines, PTFs and CAFs was confirmed by Elisa assay. n = three independent experiments, *P < 0.05 or **P < 0.01 by ANOVA. h The secretion of TSP-4 in CM-PTFs and CM-CAFs was assessed by western blot. n = 15, *P < 0.05 or **P < 0.01 by Student’s t-test. i The expression of TSP-4 was upregulated in stroma of GBC compared to that in normal tissues, as determined by IHC staining. **P < 0.01 by Student’s t-test. j Kaplan-Meier survival curves of overall survival in our GBC patients’ cohort (n = 75). Patients were assigned into two subgroups according to the median expression of TSP-4 in GBC stroma. **P < 0.01by two-sided log-rank test