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Fig. 4 | Journal of Translational Medicine

Fig. 4

From: Valtrate, an iridoid compound in Valeriana, elicits anti-glioblastoma activity through inhibition of the PDGFRA/MEK/ERK signaling pathway

Fig. 4

PDGFRA is a potential target downregulated by valtrate in GBM cells. A RNA-Seq data analysis showing the 69 intersecting downregulated genes in valtrate-treated U251 and GBM#P3 for 48 h. B Top 20 genes out of the 69 genes with the greatest log2 fold change in mRNA expression levels (basal mRNA expression level in controls of  > 1000 in U251, and  > 3000 in GBM#P3). Intersecting genes (out of the top 20) PDGFRA and FN1 are highlighted in red. C Western blot to determine expression levels of PDGFRA, FN1 and GAPDH (protein loading control) in U251, LN229 and GBM#P3 cells treated with DMSO or valtrate at the indicated concentrations for 48 h. D–E Representative images of immunofluorescence staining for PDGFRA in U251- and GBM#P3-GFP cells after treatment with the indicated concentration of valtrate for 48 h. Nuclei are stained with DAPI; GFP highlights the cells. Scale bar, 200 μm. F Analysis of expression levels of PDGFRA mRNA in normal brain tissue samples (N = 207) and GBM samples (T = 163) from the public database TCGA. *p < 0.05. G Kaplan–Meier analysis of overall survival for GBM patients with PDGFRAhigh/low GBM. PDGFRAhigh GBM, PDGFRA > median expression; PDGFRA.low GBM, PDGFRA < median expression. H–I Cell viability of U251- and GBM#P3-PDGFRA-OE under the conditions indicated, as determined with the CCK-8 assay. J–K Analysis of EdU assays for U251- and GBM#P3-PDGFRA-OE cells, showing ectopic expression of PDGFRA rescues the inhibitory effect of valtrate on the proliferation of U251 and GBM#P3 cells. L–M Percentage of apoptotic U251- and GBM#P3-PDGFRA-OE cells under valtrate treatment relative to controls. N–O Analysis of 3D spheroid invasion assays for U251- and GBM#P3-PDGFRA-OE cells under valtrate treatment relative to controls. P Western blot to determine levels of protein markers of apoptosis, invasion and migration in U251- and GBM#P3-PDGFRA-OE relative to controls under the conditions indicated. Data are shown as the mean ± SD and the differences between groups were analyzed with the Student’s t-test. n.s none significant, *p < 0.05, **p < 0.01, ***p < 0.001

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