Figure 4
From: SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations
SPARC is a key factor mediating the pro-invasive activity of S cells on M cells. (a) Validation of SPARC knockdown in S cells. Top: qPCR transcript quantification of SPARC in S cells transduced with SPARC-targeting (sh8709 and sh8711) or control shRNAs. RPL18 (S18) levels were used as internal references. Values are plotted as percent transcript levels in SPARC-knockdown vs. control cells. Bottom: Western blotting verification of SPARC knockdown by sh8709 and sh8711. Tubulin signal was used as loading and transfer reference for cell lysates, and Ponceau red staining for conditioned medium. (b) Knock down of SPARC in S cells abrogates their conditioned medium pro-invasive activity on M cells. Transwell-Matrigel assays compared the relative invasiveness of M cells treated with conditioned medium from S.shControl, S.sh8709 or S.sh8711 cells. (c) Recombinant SPARC restores the pro-invasive activity of conditioned medium from S.sh8709 cells. Transwell-Matrigel assays compared the relative invasiveness of M cells treated with conditioned medium from S.shControl, S.sh8709 or S.sh8709 cells supplemented with 20 μg/mL recombinant human (rh) SPARC. M invasiveness under rhSPARC-supplemented S.sh8709-conditioned medium was not significantly different from that induced by control medium. (d) SPARC is required for the induction of M invasiveness by co-cultured S cells. Top: S cells integrating doxycycline-inducible SPARC-specific shRNA (S.sh3399) were treated, or not, with 1 μg/mL doxycycline for 72 or 96 h and analyzed for SPARC levels by Western blotting. Tubulin was used as a loading and transfer reference. Bottom: Transwell-Matrigel assay comparing the relative invasiveness of M.GFP cells after 24 h of co-culture with control M cells or S.sh3399 cells treated, or not, with 1 μg/mL doxycycline. Invasiveness experiments in (b), (c) and (d) were performed in triplicates or quadruplicates and shown are percentages of invasiveness relative to control ± SD. Asterisks denote statistically significant differences (two-tailed Student’s t-test).