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Fig. 2 | Journal of Experimental & Clinical Cancer Research

Fig. 2

From: The U2AF2 /circRNA ARF1/miR-342–3p/ISL2 feedback loop regulates angiogenesis in glioma stem cells

Fig. 2

ISL2 transcriptionally regulates VEGFA expression and ISL2-mediated glioma conditioned-medium (GCM) regulates the proliferation, invasion, and angiogenesis of hBMECs via VEGFA mediated ERK signaling. a, b The relative expression of VEGFA after ISL2 overexpression or knockdown were detected by western blotting (a) and qPCR (b). c Secreted VEGFA levels in the GCM after ISL2 overexpression or knockdown were measured by an ELISA. d Sequence motif representing the consensus ISL2 binding motif (JASPAR database). e Schematic diagram of the putative ISL2 binding site in the 3′-UTR of VEGFA. f The luciferase reporter assays showed that ISL2 overexpression or knockdown affected the luciferase promoter activities of VEGFA. g The ChIP qPCR showed that ISL2 bound to the promoter of VEGFA. h MTS assays showed that hBMECs cell viability with ISL2 overexpression of GCM or ISL2 knockdown of GCM were reversed by additional anti-VEGFA or recombinant VEGFA, respectively. i The EDU assay showed that the proliferation of hBMECs with ISL2 overexpression of GCM or ISL2 knockdown of GCM were reversed by additional anti-VEGFA or recombinant VEGFA, respectively. Scale bar = 50 μm. j A representative Transwell assay showed the invasion of hBMECs with ISL2 overexpression of GCM or ISL2 knockdown of GCM were reversed by additional anti-VEGFA or recombinant VEGFA, respectively. Scale bar = 100 μm. k Representative tube formation assay showed that the tubulogenesis of hBMECs with ISL2 overexpression GCM or ISL2 knockdown of GCM were reversed by additional anti-VEGFA or recombinant VEGFA, respectively. Scale bar = 100 μm. l, m Western blotting showed the expression of downstream targets of the ERK signaling pathway of hBMECs with ISL2 overexpression (l) or knockdown (m) of GCM. EV: empty vector, OE: overexpression, NC: negative control, KD: knockdown. All data are expressed as the mean ± SD (three independent experiments). *p < 0.05; **p < 0.01; ***p < 0.001

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