PDGFRα GBM primary cultures demonstrate titratable and temporal DOX-inducible expression of hPDGF-A and receptor activation. PDGFRα GBM primary cultures demonstrate titratable and temporal DOX-inducible expression of hPDGF-A and receptor activation. The GBM primary cell culture (P-021) was established as described in the Materials and Methods section. (A) Cells were starved overnight in 0.1% FBS and treated with the indicated concentrations of DOX for 48 h or stimulated with exogenous recombinant PDGF-A (25 ng/ml) for 15 min. The relative amount of ligand present in these primary cultures was measured by qRT-PCR quantitation of hPDGF-A mRNA. Data are average of biological triplicate and error bars represent SD. (B) Cells were stimulated with 10 μg/ml of DOX for the indicated times, and PDGF-A mRNA expression was measured by qRT-PCR. (C) Quantitative Western blot analysis of P-021 cultures incubated with indicated concentrations of DOX for 48 h or 25 ng/ml recombinant PDGF-AA for 15 min in serum-deficient media and cell lysates probed for pTyr849 PDGFRα autophosphorylation site and total PDGFRα and control β-tubulin. Lower panel shows quantitation of Western blot (upper panel). Data are average of biological triplicate and error bars represent SD. (D) Quantitative Westerns blot analysis for temporal activation of PDGFRα activation upon acute or chronic stimulation. P-021 cells were incubated with 10 μg/mlL of DOX for the indicated times or 25 ng/ml recombinant PDGF-AA for 15 min in serum-deficient media, and cell lysates were probed and quantitated as in (C). Data represent the mean ± SD of triplicate experiments. The statistical differences were determined using paired t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure. Shuang Zhou et al. LSA 2018;1:e201800029 © 2018 zhou et al.