Fig. 1: CircPLOD2a/b are up-regulated in GBM cells under hypoxia.

A Heatmap shows differentially expressed circRNAs between normoxia and hypoxia in U87 cells on a scale from blue (downregulated, n = 2158) to red (upregulated, n = 2566). B Volcano plot shows differently expressed circRNAs between normoxia and hypoxia in U87 cells, n = 4727. Horizontal dashed line labels P = 0.05. Vertical dashed lines label |log2fold-change | = 1. C Verification of 17 up-regulated circRNAs (P-value < 0.05 and reads counts >10) by qRT-PCR, n = 3. D Basal expression levels of 9 up-regulated circRNAs in U87 cells under hypoxia examined by qRT-PCR, n = 3. E Verification of corresponding circRNAs in U87 cells by RT-PCR using divergent and convergent primers. F Schematic illustration of the generation of circPLOD2a/b. G Junction sites of circPLOD2a/b were validated by Sanger sequencing. H Relative levels of circPLOD2a/b after treated with or without RNase R digesting determined by qRT-PCR in U87 cells, n = 3. I, J Relative expression levels of (I) circPLOD2a and (J) circPLOD2b under normoxia and hypoxia in GBM cell lines determined by qRT-PCR, n = 3. K Cellular localization of circPLOD2b examined by FISH with a junction specific antisense probe (red). Scale bar: 25 μm. L U87 cells were treated for 0 h, 12 h, 24 h, 48 h under 1% O₂. Protein level of HIF1α and expression level of circPLOD2a/b were verified by western blot and qRT-PCR respectively, n = 3. M, N Protein level of HIF1α and expression level of circPLOD2a/b in (M) OE-HIF1α and (N) KD-HIF1α U87 cells under hypoxia, n = 3. Relative integrated density normalized to β-actin was marked above each band. Data are shown as mean ± SEM (error bars) and were analyzed using Student’s t-test (M) and ANOVA (C–N). NA, not available.