Figure 6.
CAF activation by cancer-derived PDGF mediates chemoresistance and HIF-1α activation of cancer cells
(A) Western blot analysis of GFP-labeled cancer cells (OVN-48) and mCherry-labeled CAFs with the indicated antibodies.
(B) Western blot analysis of CAFs, grown under monoculture or co-culture conditions for 3 days with the indicated antibodies. Note that a PDGFRB level was reduced under co-culture conditions, presumably via negative feedback regulation.25
(C) Western blot analyses of CAFs treated with 40 nM PDGFB for 3 days.
(D) Relative growth of CAFs treated with different concentrations of PDGFB for 7 days. The data are presented as mean ± SD (n = 3). p values were determined by Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001.
(E) Western blot analyses of CAFs subjected to Cas9/CRIPSR-mediated knockout with the indicated sgRNAs.
(F) Relative growth of CAFs transduced with the indicated sgRNA and then treated with 20 nM of PDGFB for 7 days. The data are presented as mean ± SD (n = 3). p values were determined by Student’s t test. Statistically significant differences are indicated. ∗∗p < 0.01.
(G) Western blot analyses of control and PDGFRB-deficient CAFs that were FACS-sorted on mCherry after incubation with cancer cells for 3 days.
(H) Survival of control and PDGFRB-deficient CAFs that were incubated with cancer cells for 7 days (n = 3). ∗∗p < 0.01, ∗∗∗p < 0.001.
(I) Western blot analysis of cancer cells that were FACS-sorted on GFP after incubation for 3 days with control or PDGFRB-deficient CAFs. p values were determined by Student’s t test. Statistically significant differences are indicated: ∗∗p < 0.01, ∗∗∗p < 0.001.
(J) Proliferation of cancer cells cultured for 7 days with control or PDGFRB-deficient CAFs in the presence of the indicated concentrations of carboplatin (n = 3). ∗∗∗p < 0.001.