Fig 1: LncRNA FOXD2-AS1 was highly expressed in glioma tissues and cells as well as GSCs. A, LncRNA FOXD2-AS1 expression predicted in GSE104267 (p = 1.482E-07). The box on the left represents expression of lncRNA FOXD2-AS1 in normal tissues and the box on the right represents expression in cancer tissues. B, A box plot displaying lncRNA FOXD2-AS1 expression in glioma samples (left) and normal samples (right) from TCGA database using GEPIA. C, Survival curve of patients with glioma regarding lncRNA FOXD2-AS1 expression from TCGA database using GEPIA (HR = 1.8, p = 0.032). D, LncRNA FOXD2-AS1 expression in glioma tissues and normal tissues detected by RT-qPCR (n = 26). E, A Kaplan–Meier one-way survival analysis according to lncRNA FOXD2-AS1 expression, followed by log-rank test (n = 26). F, FOXD2-AS1 expression in glioma cell lines determined using RT-qPCR. G, Expression of lncRNA FOXD2-AS1 and GSC markers (OCT4, SOX2, Nanog, Nestin and CD133) determined using RT-qPCR. H, Expression of Nestin and GFAP in GSCs determined by immunofluorescence. I, CD133+ GSCs selected using flow cytometry. J, LncRNA FOXD2-AS1 expression in GSCs determined using RT-qPCR. * p < 0.05. Measurement data were expressed as mean ± standard deviation. Paired t-test was used to analyse comparison within group. Unpaired t-test was adopted to analyse differences between two groups. Differences among multiple groups were analysed by one-way ANOVA, followed by a Tukey multiple comparisons post-test. Comparisons among multiple groups at different time points were performed with two-way ANOVA. Cell experiments were conducted three times independently.
Fig 2: Silencing lncRNA FOXD2-AS1 suppressed stemness and proliferation but induced apoptosis and differentiation of U251 GSCs. U251 GSCs were transfected with lncRNA FOXD2-AS1 shRNAs and sh-NC. A, Silencing efficiency of lncRNA FOXD2-AS1 shRNA in U251 GSCs determined using RT-qPCR. B, The number of main spheres every 1000 GSCs and sphere at the second passage every 100 GSCs. C, Expression of GSC markers (OCT4, SOX2, Nanog, Nestin and CD133) determined using RT-qPCR. D, CD133+ cells in U251 GSCs measured by flow cytometry. E, U251 GSC proliferation detected using colony formation in soft agar. F, U251 GSC apoptosis determined using flow cytometry. G-H, GFAP and CD133 expression in GSCs determined by immunofluorescence staining (400 ×). * p < 0.05. Measurement data were expressed as mean ± standard deviation and analysed by unpaired t-test. Cell experiments were conducted in triplicate.
Fig 3: LncRNA FOXD2-AS1 elevation induced GSC stemness and inhibited GSC differentiation via activation of the NOTCH signalling pathway via TAF-1 recruitment. GSCs were transfected with oe-NC, oe-lncRNA FOXD2-AS1 or oe-lncRNA FOXD2-AS1 + sh-TAF-1. A, Western blot analysis of expression of NOTCH pathway related genes (JAG1, PS1 and HES1). B, The number of main spheres every 1000 GSCs and sphere at the second passage every 100 GSCs. C, OCT4, SOX2, Nanog, Nestin and CD133 expression detected using RT-qPCR. D, CD133+ cells in U251 GSCs assessed by flow cytometry. E, U251 GSC proliferation measured by colony formation in soft agar. F, U251 GSC apoptosis evaluated by flow cytometry. G, GFAP and CD133 expression in GSCs assessed by immunofluorescence staining. * p < 0.05. Measurement data were expressed as mean ± standard deviation and analysed by one-way ANOVA, followed by a Tukey multiple comparisons post-test. Cell experiments were repeated in triplicate.
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