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KSHV-induced ligand mediated activation of PDGF receptor-alpha drives Kaposi's sarcomagenesis

Fig 5

PDGFR signaling drives proliferation and angiogenesis in mECK36 cells and tumors via a Rac1-ROS-NOX dependent pathway.

(A) Rac1 activation levels determined by a GTP-bound Rac1 pull-down assay/ Rac1 immunoblotting of mECK36 cells treated with PDGF-BB in the presence or in the absence of Rac1 inhibitor EHT1864. (B) ROS production (superoxide) of mECK36 cells stimulated with PDGFBB. Rac1 inhibitor (EHT1864) or NOX inhibitor (DPI) were added before PDGF stimulation (C) mRNA expression levels of c-Myc, VEGFA and KSHV LANA determined by RT-qPCR of mECK36 cells stimulated with PDGF-BB in the presence of absence of Rac1 inhibitor EHT1864, ROS scavenger NAC, or NOX inhibitor DPI. Data were from three independent experiments carried out in triplicate and are presented as means ± SD. *P < 0.05. (D) mRNA expression levels of KSHV v-Cyclin, v-FLIP, v-IL6, vGPCR and ORF8 determined by RT-qPCR of mECK36 cells stimulated with PDGF-BB. Data were from three independent experiments carried out in triplicate and are presented as means ± SD. *P < 0.05. (E) and (F) Proliferation (E) or VEGF secretion measured by ELISA (F) of mECK36 cells stimulated with PDGF-BB in the presence or absence of NAC, DPI or EHT1864. Data were from three independent experiments carried out in triplicate and are presented as means ± SD. *P < 0.05. (G) Phosphorylated and total PDGFR levels in NAC-treated and control mECK36 tumors were determined by immunoblotting. (H) mRNA levels of PDGFs and PDGFRs in NAC treated and control mECK36 tumors were determined by RT-qPCR. Data were from three tumors per treatment and are presented as means ± SD. *P < 0.05.

Fig 5

doi: https://doi.org/10.1371/journal.ppat.1007175.g005