Fig. 7.

Effect of TGFβ and linc-ROR on spheroid formation. Stromal independent growth was examined in single cells in a limiting dilution assay. (A–C) EVs were isolated from donor HepG2 cells incubated with diluent or 10 ng/ml of TGFβ for 72 h. Recipient HepG2 cells were incubated with these EVs for 48 h. Cells were then collected and plated on Ultra-Low Attachment 96 well plates with serum free DMEM medium. (D–F) HepG2 cells were transfected with either siRNA to linc-ROR-1 or non-targeting control siRNA. After 48 h, cells were collected and plated on Ultra-Low Attachment 96 well plates with DMEM medium containing 1% FBS. Spheroid formation assays were performed as described in Section 2 after 7 days. (A, D) Proportion of cells forming spheroids. (B, E) Relationship between initial cell number and average number of colonies per well. Bars express the mean value ± SEM of 12 separate determinants based on Poisson distribution. ^∗p < 0.05. (C, F) Representative photographs of spheroids at day 7 with 2000 cells/well incubated with EVs isolated from cells incubated with diluent or 10 ng/ml TGFβ, or transfected with control siRNA or siRNA to linc-ROR-1. Bar represents 100 μm.