Fig 2: Effects of GADD45A on cell viability and apoptosis of CMEC via the JNK/p38 MAPK signaling pathway. (A) Cell viability was determined by Cell Counting Kit-8 assay in CMECs. (B and C) Apoptotic rates were determined by flow cytometry. (D) Protein expression levels of BCL2, Bax, cleaved caspase 3 and caspase 3 were determined by western blotting analysis. Each experiment was repeated at least three times. *P<0.05, **P<0.01, ***P<0.001. CMEC, cardiac microvascular endothelial cells; GADD45A, growth arrest and DNA damage-inducible α; H/R, hypoxia/reoxygenation; NC, negative control; siRNA, small interfering RNA.

Fig 3: LINC01116 mediates the MDM2-p53 pathway in glioma. (A) Gene Expression Profiling Interactive Analysis revealed that LINC01116 levels negatively correlate with the mRNA levels of p53 target genes BAK1, BAX, CDKN1A and GADD45A in GBM tissues (n=81). (B and C) LINC01116 regulated the mRNA expression of MDM2, BAK1 and GADD45A, but not TP53 in glioma cell lines. (D) Protein expression of MDM2, p53, BAK1 and GADD45A were regulated by LINC01116 in glioma cells. (E) Histogram indicating the relative protein levels in (D). (F) LINC01116 regulated the p53 pathway in A172 cells, which was dependent on the presence of MDM2. (G) Histogram indicating the relative protein levels in (F). *P<0.05, **P<0.01 and ***P<0.001 vs NC. LINC01116, long intergenic non-protein coding RNA 01116; MDM2, E3 ubiquitin-protein ligase Mdm2; KD, knockdown; OE, overexpression; NC, negative control; CDKN1A, cyclin-dependent kinase inhibitor 1; GADD45A, growth arrest and DNA damage-inducible protein GADD45 α.

Fig 4: LINC01116 promotes gliomagenesis in vivo. (A) LINC01116 KD inhibited the growth of Shg44 cell tumors. Histogram indicating the mean weight of the xenograft tumors (n=5). Line chart of tumor growth curves (n=5). (B) mRNA expression levels of LINC01116, miR-744-5p, MDM2 and TP53 in the indicated xenograft tumors. (C) Protein expression of BAK1, GADD45A, MDM2 and p53 in xenograft tumors. (D) TUNEL assays were performed to detect glioma cell apoptosis in the xenograft tumors. Histogram indicating the percentage of apoptotic cells in the xenograft tumors (n=5). Magnification, ×20. **P<0.01 and ***P<0.001 vs NC. LINC01116, long intergenic non-protein coding RNA 01116; miR, microRNA; KD, knockdown; NC, negative control; GADD45A, growth arrest and DNA damage-inducible protein GADD45 α; MDM2, E3 ubiquitin-protein ligase Mdm2.

Fig 5: NMD inhibition restores the p53 pathway and triggers p53β/γ expression in p53 mutant cancer cells.A, schematic showing the location of premature termination codons in p53 mutant cell lines. B, mRNA expression FC of mutant p53α in cells treated with NMDi. C, Western blots showing increased expression of truncated p53 upon NMDi treatment in p53 mutant cells. D, Western blot quantification of mutant p53 protein expression shown in (C), in indicated cells treated with NMDi (E and F) mRNA expression FC of p53β and p53γ isoforms, respectively, in NMDi-treated p53 mutant cells. G–I, mRNA expression FC of p53 transcriptional targets GADD45A, p21, and PUMA, respectively, upon NMDi treatment. J, Western blots showing increased expression of p21 and PUMA in NMDi-treated cell lines shown. K, Western blot quantification of p21 and PUMA protein expression in indicated cells treated with NMDi. GAPDH or vinculin were used as loading controls for Western blots. For H2228, Western blot from (C) was reprobed with p21 in (J). For TCCSUP and UACC-893, two sets of same samples were run side by side on the same gel; one set was probed with p53 and PUMA, another set was probed with p21 and Vinculin. Cells in B–J were treated with either dimethyl sulfoxide (Control) or 1 μM NMDi for 16 h. RT-qPCR data shown (n = 4–6) and Western blot quantifications are from three independent experiments. Mean ± SD, p values, two-tailed t tests, ∗ ≤0.05, ∗∗ <0.01, ∗∗∗ <0.001, ∗∗∗∗ <0.0001, ∗∗∗∗∗ <0.00001, NS, not significant. FC, fold change, NMD, nonsense-mediated decay; NMDi, NMD inhibitor.