Fig 2: The combination treatment with p75IgG-Sap enhances entinostat cytotoxicity in neuroblastoma multicell spheroids. (A) A total of 24 h after cell seeding, SH-SY5Y multicell spheroids (mcs) were preincubated for 24 h with either vehicle or 1 µM entinostat (entin). Thereafter (time 0), the spheroids were incubated for the indicated time periods with either vehicle, 30 nM p75IgG-Sap, 1 µM entinostat, or the combination of entinostat plus p75IgG-Sap. Digital images were acquired by light microscopy at the indicated time points. The mcs size of each experimental group is reported as percent of the control (vehicle at time 0). Scale bar = 200 µm. Values are the mean ± SD of four independent experiments. *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. entinostat alone by ANOVA followed by Bonferroni’s test. (B) SH-SY5Y spheroids that were grown for six days were pretreated for 48 h with either vehicle or 1 µM entinostat and the exposed for 72 h to either the vehicle or 30 nM p75IgG-Sap. At the end of the incubation, digital images were acquired by light microscopy. The mcs size of each experimental group is reported as percent of the control (vehicle + vehicle). Scale bar = 200 µm. (C) SH-SY5Y spheroids that were grown for six days and treated as in (B) were analyzed for cleaved PARP by Western blot. Values are the mean ± SD of four independent experiments. ** p < 0.01, *** p < 0.001 vs. vehicle. ## p < 0.01, ### p < 0.001 by ANOVA followed by Tukey’s test.

Fig 3: DCZ0415 suppressed CRC cell proliferation, invasion as well as migration, promoted cell apoptosis, and resulted in the arrest of cell cycle.A DCZ0415 decreased the expression of TRIP13 in CRC cells. B DCZ0415 inhibited cell proliferation of CRC cells. C DCZ0415 significantly reduced number of cell colonies. D DCZ0415 suppressed invasion and migration of CRC cells. E DCZ0415 increased apoptosis of CRC cells. F DCZ0415 induced G0/G1 phase arrest of CRC cells. G The protein level of PCNA, Bcl-2, Cdk4, Cyclin D1, MMP2, MMP9, Vimentin, N-cadherin, p-EGFR, and p-AKT was downregulated, whereas E-cadherin, Bax, cleaved PARP, and caspase3 level were upregulated. *p < 0.05, **p < 0.01; ns, no significant difference.

Fig 4: miR-422a inhibition attenuates OGD/R-induced neuronal cell death and apoptosis.SH-SY5Y cells were infected with pre-miR-422a anti-sense lentivirus (“lv-antagomiR-422a”) or non-sense miR inhibitor control lentivirus (“lv-antagomiR-C”) for 24 h, followed by puromycin selection (4–5 passages) to establish stable cells. Cells were maintained under oxygen glucose deprivation (OGD) for 4 h, followed by re-oxygenation (“OGD/R”) for the applied time; miR-422a expression (a), cell viability (b), cell death (c), cleaved-/or total-caspase-3/-PARP expression (d) and cell apoptosis (e) were tested by the assays mentioned in the text, with mitochondrial depolarization examined by JC-1 staining assay (f). The primary murine neurons were transfected with non-sense miR inhibitor control (“antagomiR-C”, 100 pmol × two rounds) or miR-422a inhibitor oligonucleotides (“antagomiR-422a”, 100 pmol × two rounds), followed by the same OGD/R procedure for applied time; miR-422a expression (g) and cell death (medium LDH release, h) were tested; Cell apoptosis and mitochondrial depolarization were tested by TUNEL staining (i) and JC-1 assay (j), respectively. Data indicate standard deviation (SD, n = 5). *p < 0.05 vs. “Mock” cells. #p < 0.05 vs. OGD/R-treated “lv-antagomiR-C”/“antagomiR-C” neuronal cells. Each experiment was repeated three times and similar results were obtained. Bar = 100 µm (f, i and j).

Fig 5: Sevoflurane induces neural apoptosis in the hippocampus and temporal lobe. a Representative immunoblots showing the protein expression of Bax, Bcl-2, cleaved caspase-3 and cleaved PARP in the hippocampus and temporal lobe of young mice 24 h after the last exposure to sevoflurane. n = 4 for each group. b, c, d, e Histograms showing the results of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP blots in the hippocampus and temporal lobe. *P < 0.05 versus the Con group. In the temporal lobe, #P < 0.05 versus the Con group. f Representative images showing the flow cytometry analysis of the apoptosis ratio in PC12 cells 24 h after the last exposure to sevoflurane. Early apoptotic populations (PE+7-AAD-cells) are in the lower-right quadrant; late apoptotic cells (PE+7-AAD+) are in the upper-right quadrant in each dot plot. g Representative quantification of the flow cytometry analysis of the apoptosis ratio of PC12 cells 24 h after the last exposure to sevoflurane. h Representative CCK-8 assay showing cell viability in primary hippocampal neurons 24 h after the last exposure to sevoflurane. n = 5 for each group. *P < 0.05 versus the Con group. #P < 0.05 versus the LV5-GLUT3 group. The data are presented as the mean ± s.e.m