Fig 2: 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 3: Mitochondria internalization altered apoptosis rate and the expression of apoptosis related proteins in post-ODG VSC4.1 motor neurons. (A-B) Post-OGD VSC4.1 motor neurons were co-incubated with mitochondria for 24 h. The apoptosis rate of neurons were analyzed by FACS analysis. Each data point is presented as mean ± SEM. **P < 0.01, vs normal group; #P < 0.05, vs OGD group, n=8. (C) Expression levels of Bcl-2, Bak, GRP78, CHOP and P-AKT were analyzed by western blot assay in the Control, OGD+Vehicle and OGD+Mito groups. (D-G) Quantification of the optical densities of the protein to determine the levels of protein expressed as percentages of control values. The data are presented as mean ± SEM from three independent experiments. **P< 0.01, vs control group; #P < 0.05 vs OGD+Vehicle group.

Fig 4: Schematicview of apoptosis by nintedanib. Both intrinsic and extrinsic apoptotic regulators are dysregulated in mesenchymal cells of fibrotic lungs. Nintedanib induces apoptosis by increasing the expression of pro-apoptotic genes Bak and Bax.

Fig 5: MCL-1 dependence requires BAX/BAK.A Diagram of experimental set-up for results presented in B. MMTV-PyMT CRISPR/Cas9 Bax/Bak cell line (derivation described in Supplementary Fig. 2A) was injected into mammary fat pad of FVB recipients and tumours allowed to grow to ~5 mm before assignment to vehicle or S63845 treatment groups. S63845 treatment was at 25 mg/kg IV twice per week for 3 weeks. B Mean tumour weight of mice described in A, following 3 weeks of vehicle control (n = 7) or S63845 (n = 7) treatment (25 mg/kg IV twice per week) is shown (±SD), n.s. not significant unpaired t test. C Diagram of experimental set-up for results presented in D–F. MMTV-PyMT; CRISPR/Cas9 Bax/Bak tumour cell line (as used in A, B, this cell line carries RosaCRE-ERT2;Mcl1fl/fl alleles that were not activated in A, B, derivation described in Supplementary Fig. 2A) was injected into mammary fat pad of FVB recipients and tumours allowed to grow to ~5 mm before assignment to vehicle or tamoxifen groups (tamoxifen induces CRE-ERT2-mediated deletion of Mcl1fl/fl); therefore, vehicle treated tumours will be MCL-1 proficient and tamoxifen-induced tumours (TAM) will be MCL-1 deficient. Tumour size was monitored by calliper measurement thrice weekly until harvesting at predefined ethical endpoint (15 mm diameter). D Points indicate individual tumour weights at endpoint, vehicle control (n = 5) or tamoxifen (n = 5). Error bars represent mean ± SD, n.s. not significant unpaired t test. E Kaplan–Meier graph showing tumour-related survival of mice shown in D. Day 0 indicates vehicle/tamoxifen induction (at tumour diameter ~5 mm). Mcl1 deletion in established tumours does not alter survival when Bax/Bak have been CRISPR/Cas9 targeted. Vehicle control (n = 5) or tamoxifen (n = 5). P = 0.2482 Log-rank (Mantel–Cox) test. F Western blot quantification shows sustained decrease in MCL-1 from endpoint tumours shown in D, E. Tumour material sampled for western blot will include host-derived (WT for Mcl1) stroma and donor-derived tumour epithelium. MCL-1 expression is calculated relative to β-actin control for each sample, each point represents an individual tumour and error bars represent mean ± SD. **P < 0.01, unpaired t test. Western blots shown in Supplementary Fig. 3B.