Fig 1: Effect of AAT on Mcl-1 phosphorylation, the activity of MAP kinases and caspases.Neutrophils from healthy volunteers (2.5 × 106/ml) were cultured in medium supplemented with patient serum (3 mg protein/ml; 1%) and those containing low levels of AAT (AAT-reduce serum; 1%) in the presence of STS (0.2 µM). After 3 h, the expression of pMcl-1 (A, n = 7), pAkt (B, n = 8) and pERK1/2 (C, n = 10) were analyzed by western blot. Expression levels of the phosphorylated proteins were normalized to that of the unphosphorylated forms. GAPDH was used as loading control. One representative blot is displayed. *p<0.05; **p<0.01; n.s. = not significant. D. After 4 h of incubation the activities of caspase-9 and caspase-3/-7 were quantified. Results are presented as means ± SEM of eight independent experiments. No significant differences were found (one-way ANOVA with Newman keuls post-hoc test).
Fig 2: SERPINA1 is upregulated in glioma. (a) Differential expression of SERPINA1 in glioblastoma (box plot) (red: tumor tissue, gray: normal tissue). (b and c) Kaplan–Meier survival curves of disease-free survival and overall survival of patients who had gliomas with differential expression of SERPINA1. (d and e) qRT-PCR analysis of SERPINA1 mRNA expression and western blotting analysis of SERPINA1 protein in NHA, U251, T98G, LN-229, and A172 cell lines. One-way ANOVA was conducted. (f) Co-staining of SERPINA1 (red) with DAPI (blue) in NHA, T98G, and A172 cell lines. One-way ANOVA was conducted. Data are expressed as means ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001, n = 3).
Fig 3: NQO1 binds to the 3'UTR of SERPINA1 mRNA and suppresses its translation, and SERPINA1 rescues effects of sh-NQO1 on proliferation and apoptosis of primary glioma cells. (a) Expression of SERPINA1 in sh-NQO1#1/2 transfected primary glioma cells was assessed by qRT-PCR. One-way ANOVA was conducted. (b) SERPINA1 protein levels in sh-NQO1#1/2 transfected primary glioma cells. One-way ANOVA was conducted. (c) The ratios of FL/RL activity in sh-NQO1 transfected primary glioma cells. Student’s t test was conducted. (d) Colony formation assay results and quantitative analysis of primary glioma cells after transfection with sh-NQO1, pcDNA-SERPINA1, sh-NQO1 + pcDNA-SERPINA1. One-way ANOVA was conducted. (e) Annexin V/7AAD double staining assay results and quantitative analysis of primary glioma cells after indicated transfections. One-way ANOVA was conducted. Data are expressed as means ± SEM (**p < 0.01, ***p < 0.001 vs sh-NC or control, ### p < 0.001 vs sh-NQO1, n = 3).
Fig 4: Serum AAT confers resistance to STS in human neutrophils.A. Neutrophils from healthy volunteers (2 × 106/ml) were cultured in medium supplemented with different concentrations of plasma-derived human AAT (range 0–2 mg/ml) and treated with STS (0.2 µM). After 18 h, apoptosis was quantified by propidium iodide staining and FACS analysis. Percentage of apoptotic cells displaying fragmented DNA is depicted. Results are presented as mean ± SEM of three independent experiments. *p<0.05 (t-test). B. Neutrophils cultured in medium supplemented with the albumin-depleted fraction B11 and the serum fraction depleted for Ig for 9 times (3%), which have been shown to not confer resistance to STS, were additionally pre-incubated with 2 mg/ml plasma-derived AAT. After 1 h, STS (0.2 µM) was added and cells were further cultured overnight. Data of three independent experiments are expressed as fold changes vs. control cells cultured in the presence of patient serum. No significant differences could be found. C. Pooled patient serum was first diluted to a final concentration of 3 mg protein/ml. Then, serum was reduced for AAT by using an AAT-specific antibody and AAT decrease was further confirmed by Elisa. Empty beads were used as control. AAT concentrations are depicted in the table on the right. Patient serum, AAT-reduced serum (each 1%) with or without plasma-derived human AAT (native AAT; 2 mg/ml) were added to the culture medium of neutrophils (2 × 106/ml). After treatment with STS (0.2 µM) for 18 h, apoptosis rate was quantified. Fold changes in apoptosis vs. control cells are displayed. Graphs show results of four independent experiments. **p<0.01 vs. untreated control; §p<0.05 (one-way ANOVA with Newman keuls post-hoc test).
Fig 5: Verification of the differences observed with TMT labeling by western blotting. HPT and A1AT were analyzed by western blotting to confirm the reliability of SRM. * and ** showed statistical significance, *p < 0.05, **p < 0.01. A1AT, alpha-1-antitrypsin; RI, relative intensity.
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