Fig 2: Sevoflurane promotes caspase-1-dependent pyroptosis in BV2 microglia (A). Cleaved caspase-1 and caspase-3, IL-1β, and IL-18 levels were detected by Western blotting analysis in BV2 microglia exposed to 4% sevoflurane for 6 h (B). Western blotting analysis of caspase-1, caspase-3, IL-1β, and IL-18 induced by sevoflurane in BV2 cells pretreated with 100 ng/ml of lipopolysaccharide (LPS). (C) Pyroptosis-related proteins (gasdermin D (GSDMD) and gasdermin E (GSDME)) were detected by Western blotting analysis induced by sevoflurane in BV2 cells. (D) Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays of cell viability and LDH activity in BV2 cells. (E). FAM-FLICA caspase-1 assays of cleaved caspase-1 (green), propidium iodide (PI) (red), and DAPI (blue) in BV2 cells. Scale bar: 50 μm. Cleaved caspase-1 fluorescence intensity was calculated as the iod. (F). FAM-FLICA caspase-1 assays of cleaved caspase-1 (green) in BV2 cells. Scale bar: 50 μm. ***p < 0.001, **p < 0.01, *p < 0.05.

Fig 3: LNT enhances the sensitivity of EC-109 cells to Oxa. (a) The conjunction of LNT and Oxa greatly reduced EC-109 cell growth (P < 0.05). (b) The combination of LNT and Oxa persuaded programmed cell death in EC-109 cells (P < 0.05). (c) LNT combination and Oxa induced mRNA expression levels of BAX and CASP3 and inhibited the mRNA expression BCL2 (P < 0.05) level. (d) The conjunction of LNT and Oxa increased BAX and cleaved caspase 3 protein expression while suppressing BCL2 protein expression (P 0.05). (e) The analysis of the combination index (CI) of LNT and Oxa using the CompuSyn software revealed that both LNT and Oxa acted synergistically on EC-109 cells. The data were expressed as the mean standard deviation for each experiment, which was done in triplicate. P < 0.05 in comparison to 0 g to analyze the differences between groups; statistical analysis using one-way analysis of variance (ANOVA) was used.

Fig 4: IHC analysis of neuron protection and cell apoptosis in the perilesional zone. (A) Example sections showing IHC staining of NeuN (indicating mature neurons) and caspase-3 (indicating cell apoptosis). Fields from the perilesional zone with the highest positive cell count were selected (magnification, ×200). (B) Results of staining analysis. IHC, immunohistochemical; TBI, traumatic brain injury; TMS, transcranial magnetic stimulation.

Fig 5: The binding of ribosomal protein to UNC5B truncates was closely related to cell proliferation and tumour formation in mice. A–D, PPI analysis was shown by overexpressing different intracellular domains of UNC5B (A, C: residues 399‐945, B, D: residues 412‐945) in 5637 and T24 cells, respectively. Ribosomal proteins aggregated into complexes were marked dark blue. E, WB analysis demonstrated that S6 ribosomal protein was increased by transfecting UNC5B truncates and full‐length UNC5B. The expression of cleaved PARP was elevated in UN‐5637 and UN‐T24 cells. However, no band corresponding to cleaved caspase‐3 was found in both groups of cells. Moreover, there was no change in the expression of P53 or mutant P53. F, S6 ribosome protein and BCL‐2 were co‐immunoprecipitated by CO‐IP tests. Cleaved caspase‐3 was seldomly co‐immunoprecipitated in 5637 group of cells, but not in T24 group. Co‐IP tests also revealed that the overexpression of UNC5B and UNC5B truncates increased the binding of S6 ribosomal protein. More binding of BCL‐2 was also found in 399′5637, UN‐5637, 399′T24 and UN‐T24 cells, but not in 412′T24 cells. G, The normalization of the binding of UNC5B to S6 ribosome protein was shown in CO‐IP tests (top: 5637 cells; below: T24 cells). H, 399′5637 and 412′5637 cells, UN‐5637 and NC‐5637 cells were separately injected into the bilateral armpits of mice of two groups. The formation of tumours was observed 56 days after the injection. I, Image of tumour volume was shown after the injection of the different cells. The formula (volume = length*width2/2)was applied to estimate the tumour size. Student's t tests. *P < .05. **P < .01 (mean ± SD, n = 5)