Fig 2: MYST1 is correlated with the prognosis of patients with glioma. A, The analysis of TCGA data of MYST1 mRNA expression in GBM and normal tissues were obtained and analyzed in the GEPIA. B, The analysis of MYST1 mRNA expression in gliomas with CIC mutation from the database termed Tumor Glioma (CIC mutation status) Gleize‐30‐MAS5.0‐u133p2. C, The analysis of MYST1 mRNA expression in the primary and recurrent GBM in the data acquired from the GlioVis. D, The relative mRNA level and protein level of MYST1 in normal HEB gliocytes (N) and primary cells derived from different grades of gliomas. G1T, grade I gliomas; G3T, grade III gliomas; G4T, grade IV gliomas. E, MYST1 gene methylation levels of different WHO grades of gliomas in the CGGA methyl‐159 datasheet. F and G, Kaplan‐Meier (KM) analysis and Log‐rank test demonstrated that the elevated MYST1 protein expression in glioma is correlated with shorter overall survival in two connected databases titled with Tumor Glioma‐IGS (Core‐Transcript) French‐95‐rma_sketch‐huex10t and Tumor Glioma‐IGS (Core‐Exon) French‐95‐rma_sketch‐huex10p from the R2 microarray analysis and visualization platform. H, KM analysis and Log‐rank test of MYST1 expression data in different subtypes from Vital GBM datasheet downloaded from GlioVis. I, KM analysis and Log‐rank test of MYST1 expression data in WHO grade II gliomas in the datasheet downloaded from the CGGA. J, KM analysis and Log‐rank test of MYST1 methylation level in gliomas in the datasheet downloaded from the CGGA. Experimental data were used as mean ± SD, n ≥ 3, significant difference was tested by Student's t test. *P < .05, **P < .01, ***P < .001, ****P < .0001. P < .05 was considered as statistically significant. GBM, glioblastoma; GEPIA, Gene Expressing Profiling Interactive Analysis

Fig 3: Inhibition of EGFR by erlotinib recovered GBM progression induced by MYST1 overexpression. A, MTT assay to detect cell proliferation in LN229 and U87 cells with MYST1/GFP overexpression after erlotinib (200 ng/mL)/DMSO treatment (n = 3). B, Western blots of proteins expression in EGFR signaling pathways in LN229 and U87 cells with MYST1/GFP overexpression after erlotinib (200 ng/mL)/DMSO treatment. All data were used as mean ± SD, n ≥ 3, significant difference was tested by Student's t test. **P < .01, ***P < .001. P < .05 was considered as statistically significant. EGFR, epidermal growth factor receptor; GBM, glioblastoma; GFP, green fluorescent protein

Fig 4: MYST1 overexpression promotes GBM progression and upregulates EGFR signaling pathway. A, Western blots of proteins expression in EGFR signaling pathways in LN229 and U87 cells after MYST1/GFP overexpression. B, qRT‐PCR analysis of EGFR mRNA expression after MYST1 overexpression. C, MTT assay reveals significant cell proliferation promotion induced by MYST1/GFP overexpression in LN229 and U87 cells (n = 3). D and E, The effect of MYST1/GFP silencing on tumorigenicity in BALB/c‐nu nude mice. LN229 and U87 cells with MYST1/GFP overexpression were injected subcutaneously into the nude mice (n = 4). Tumor burden was monitored every 4 d by a digital caliper. Tumor size was calculated as the formula: length × width2 × π/6. After the experiment, mice were sacrificed and the tumor was taken out and weighted. F, Western blots of proteins expression in EGFR signaling pathways in indicated tumors obtained from the nude mice. All data were used as mean ± SD, n ≥ 3, significant difference was tested by STUDENT's t test. **P < .01, ***P < .001. P < .05 was considered as statistically significant. EGFR, epidermal growth factor receptor; GBM, glioblastoma; GFP, green fluorescent protein

Fig 5: MYST1 silencing inhibits cell proliferation in GBM cells. A, Quantitative real‐time PCR (qRT‐PCR) and western blot analysis of MYST1 mRNA and protein expression in several GBM cell lines. B, qRT‐PCR and Western blot analysis of MYST1 mRNA and protein expression in LN229, U87 and A172 cells after MYST1/GFP silencing. C, MTT assay reveals significant cell proliferation inhibition induced by MYST1 silencing in LN229, U87 and A172 cells, compared to that of the GFP silencing group (n = 3). D, Cell counting by trypan blue assay reveals significant cell proliferation inhibition induced by MYST1 silencing in LN229, U87 and A172 cells, compared to that of the GFP silencing group (n = 3). E, BrdU‐positive cells in LN229, U87 and A172 after MYST1/GFP silencing. All data were used as mean ± SD, n ≥ 3, significant difference was tested by Student's t test. *P < .05, **P < .01, ***P < .001. P < .05 was considered as statistically significant. GBM, glioblastoma; GFP, green fluorescent protein