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. 2021 Sep 2;22(5):1246. doi: 10.3892/etm.2021.10681

Figure 3.

Figure 3

(A) The expression of p53 in glioblastoma cell lines SHG44 and U87 was significantly upregulated following TMZ treatment. In the cell immunofluorescence assay, p53, nucleus and mitochondria were stained green, blue and red, respectively, which indicated that the upregulated p53 predominantly accumulated in the nucleus following TMZ treatment (scale bar, 80 µm). (B) Following treatment with TMZ, the upregulation of p53 and the activation of its downstream apoptotic pathways mediated by the Bcl-2 family was observed. (C) In GBM cells stimulated by TMZ, downregulation of PINK1 was observed, as well as a decreased level of Ser-65 site phosphorylated ubiquitin that indicated the impaired function of PINK1. (D) Following treatment with TMZ, the co-localization of lysosome (green) and mitochondria (red) decreased, which indicated the downregulation of mitophagy. The nucleus was stained blue (scale bar, 80 µm). (E) In the p53 mutant U251 cells, TMZ treatment increased the level of p53 and PINK1. (F) A total of 62.47% cells of the control group were included in the R3 gate, while 66.78% of the TMZ treatment group and 89.63% of the TMZ + G-TPP group were in the same R3 gate. The level of ROS in GBM cells in the TMZ treatment group was not statistically different from that in the control group (P=0.2892), while the level of ROS in the G-TPP and TMZ combined treatment group was significantly higher than the TMZ group (P=0.0041). (G) Following TMZ treatment, the level of mitochondrial fusion of glioblastoma cells was increased. The mean size of the MS was elevated in SHG44 and U87 cells, and the mean ML in U87 cells was also increased, although the difference in the number of N/I was not significantly different between the two groups (scale bar, 80 µm). *P<0.05; **P<0.01; ***P<0.001. GBM, glioblastoma multiforme; G-TPP, Gamitrinib triphenylphosphonium; TMZ, temozolomide; CI, combination index; TRAP1, TNF receptor-associated protein 1; PINK1, PTEN-induced kinase 1; p, phosphorylated; Ubi, ubiquitin; ROS, reactive oxygen species. MS, mitochondrial network; ML, length of mitochondria; N/I, mitochondrial nets/individual mitochondria.