Figure 6

CECR1 activation in THP1 macrophages promotes paracrine activation of periostin protein production in pericytes. (a and b) Confocal images of imunohistological stainings of periostin, PDGFRβ and CD163 in human GBM sections. Scale bar, 50 μm. (c) Correlation between CD163 and periostin mRNA levels in a set of 154 TCGA data set-derived GBM samples. (d) QPCR analysis of periostin mRNA expression levels normalized to housekeeping genes in HUVECs, pericytes, U87, U251 and THP1 macrophages from three experiments. ***P<0.001 (pericytes versus other cell types). (e) Diagram showing experimental setup with pericytes (green) co-cultured with macrophages without (light blue) and with U87 pre-stimulation (dark blue) seeded in filter insert. Western blot shows periostin and β-actin protein levels in pericytes of the different treatment groups. (f) Quantitative results of western blot analysis normalized to β-actin loading control from four experiments. *P<0.05; ***P<0.005. (g) Correlation between periostin and CECR1 mRNA levels in a set of 205 TCGA data set-derived GBM samples. (h) Western blot shows periostin and β-actin protein levels in pericytes treated with no macrophages, with non-transfected macrophages (control), and macrophages transfected with sisham, siCECR1 or siPDGFRβ. (i) Quantitative results of western blot analysis normalized to β-actin loading control from four experiments. *P<0.05. (j) Western blot shows periostin and β-actin protein levels in pericytes treated with macrophages with and without pre-treatment with rhCECR1. Graph shows quantitative results of western blot analysis normalized to β-actin loading control of at least four experiments. *P<0.05 versus no rhCECR1 stimulation. (k) Western blot shows periostin and β-actin protein levels in pericytes treated with different concentrations of PDGFB. (l) Quantitative results of western blot analysis normalized to β-actin loading control from at least three experiments. **P<0.01; ***P<0.005 versus no PDGFB stimulation. (m) Representative western blot of three independent experiments, showing periostin and β-actin protein levels in pericytes treated with rhCECR1. (n) Western blot of periostin protein and β-actin loading control in pericytes with siPOSTN treatment, sisham or no treatment blot represents results from three observations. (o) Panel of fluorescent images of dsRed-marked pericytes that have migrated through the transwell setting in response to the different conditions (in response to supernatant of non-treated macrophages (control), or sisham- or siperiostin-treated macrophages), with and without PDGFB stimulation (scale bar, 50 μm). (p) Quantified results of the transwell migration assay from three experiments. **P<0.01 (siPOSTN versus PDGFB+siPOSTN); ***P<0.005 (sisham versus siPOSTN; control versus PDGFB+control; sisham versus PDGFB+sisham).