Fig 1: Effects of Lf and HI on protein expression in the right cortex at P4 (24 h) after injury. Dams were fed control diet (Sham and HI), Lf 0.1 (HILf0.1), 1 (HILf1), or 10 (HILf10) g/kg lactoferrin. All proteins were quantified by densitometry, and then normalised to actin or tubulin and expressed as a value (ODI) relative to the Sham group for: fractin (a), cleaved caspase 3 (b), IL-1ß (c), TLR4 (d), GFAP (e), AQP4 (f), GLT1 (g), iNOS (h), and synaptophysin (i). Representative bands of proteins with the respective molecular weight (mw) in KDa (j). All values are presented as the mean ± SEM, n = 4–6 animals per group. * HI vs. SH, $ difference between HILf groups. Differences were determined by one-way ANOVA and considered significant when p < 0.05.
Fig 2: GLP regulated the expression of cell apoptosis and autophagy. (A) Western blot analysis of the representative proteins; (B) quantification of the expression of C-Caspase 3/Caspase 3, Bcl-2/Bax, NRP-1, P62, LC-3. GAPDH was used as a control. Data are present as mean ± SD (n = 10). ##p < 0.01 as compared to Sham; *p < 0.05, **p < 0.01 as compared to Model.
Fig 3: YY-moxi alone and YY-moxi combined with NAC significantly downregulated NF-κB/p65 levels, increased Bcl-2, and decreased Bax and Caspase-3 levels in MCAO model rats. (A) The levels of Bax, Bcl-2, Caspase-3, and p56 were assessed by IHC assays in the brain tissue of each group. Magnification, 40×; scale bar =50 µm. (B) The positive areas of Bax, Bcl-2, Caspase-3, and p56 were quantitatively analyzed according to the IHC results. (C) Protein expression of Bax, Bcl-2, Caspase-3, and p56 in the injured brain tissue of each group. (D) Western blot analyses of the apoptosis-associated in the MCAO model rats. *, P<0.05, **, P<0.01 vs. the MCAO group; ##, P<0.01 vs. the YY-moxi group. IHC, immunohistochemical; MCAO, middle cerebral artery occlusion model; NAC, N-acetylcysteine; YY-moxi, YaoYi-moxibustion.
Fig 4: Radiation induced cell cycle arrest, autophagy and apoptotic cell death. a Histogram of DNA content in cells reflecting the G0/1, S, and G2/M phases of the cell cycle at 72 h post-irradiation. Stacked column graph shows mean percentage relative (%) ± SEM of triplicates from three independent experiments. b Histogram of autophagosome marker LC3B intensity in indicating the induction of autophagy following irradiation at 72 h post-irradiation. Grey overlay shows the profile at 0 Gy. Column graph shows mean ratio of autophagy induction relative to 0 Gy ± SEM of duplicates from three independent experiments. c Immunoblot of p62, LC3-I and II, and reference protein ß-actin at 72 h post-irradiation. Band intensity was data standardised to ß-actin per sample and then expressed relative to 0 Gy. Raw data is provided in Additional file 4: Fig. S2. Column graph shows mean fluorescent intensity relative to 0 Gy ± SD of from three independent experiments. d Dot plots of apoptotic marker annexin V and viability marker 7-AAD at 72 h post-irradiation. e Dot plots of apoptotic marker caspase 3/7 and viability marker 7-AAD at 72 h post-irradiation. Column and stacked column graphs show mean percentage (%) ± SEM of duplicates from three independent experiments. f Immunoblot of caspase 3 and reference protein ß-actin at 72 h post-irradiation. Band intensity was data standardised to ß-actin per sample and then expressed relative to 0 Gy. Raw data is provided in Additional file 4: Fig. S2. Column graph shows mean fluorescent intensity relative to 0 Gy ± SD of from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs 0 Gy determined by two-way ANOVA with Dunnett’s multiple comparison test
Fig 5: Risperidone causes an inhibition of autophagy and a promotion of apoptosis in femur tissues of mice. Mice were treated with 0, 0.3, 0.45, 0.6, 0.75 mg/kg of Risperidone. A representative micro-CT 3D and plan scan images of cross-sectional area of femur tissues. B statistical analysis of BMD in femur tissues of mice assessed by micro-CT. C statistical analysis of BMC in femur tissues of mice. D representative images of HE staining in femur tissues of mice. E Western blot analysis of autophagy-related proteins (LC3 II/I, Beclin1, and p62) expression in femur tissues of mice. The band intensity was assessed. F Western blot analysis of apoptosis-related proteins (cleaved PARP1, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9). Different numbers represent different concentrations of Risperidone (mg/kg). G co-localization of LC3 II (red), LC3 I (red), Beclin1 (red), and p62 (red) with the osteoblast marker RUNX2 (green) by double-labeled immunofluorescence, wherein the nucleus is labeled by DAPI (blue). *p < 0.05, compared with the treatment of 0 mg/kg of Risperidone. The experiments are repeated 3 times
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