P188 seals the membrane rupture of HT22 cell.

<p>Cells stained with PI indicated cells with disrupted membranes (PI⁺, A and D). Cells that remained permeable after P188 or PBS treatment showed green fluorescence (SYTOX® Green⁺, B and E ) or yellow in merged photograph (C and F ). Cell membrane resealed by P188 still showed red fluorescence in merged photograph but no green fluorescence (F).</p

P188 reduces ischemia/reperfusion-induced brain injury.

<p>(A) TTC staining of infarct brain regions. (B) Quantitative analysis of brain infarct volume, presented as percentage of the ipsilateral hemisphere. (C) Motor deficits and (D) brain edema. Data are represented as mean ± SD (n = 10 ). *, p<0.05 <i>vs</i> the saline-treated group. ^##, p<0.01 <i>vs</i> the sham-operated group. P188 (S) = 0.2 g/kg; P188 (M) = 0.4 g/kg; P188 (L) = 0.8 g/kg.</p

P188 attenuates BBB damage and inhibited protein levels and activity of MMP-9.

<p>(A) Evans blue dye extravasation 24 h after tMCAO. The blue staining denoted the areas with increased BBB permeability. (B) Quantification of Evans blue dye extravasation after tMCAO. Bars represent mean ± SE. *, p<0.05 <i>vs</i> saline-treated mice (n = 4). (C) MMP-9 activity analyzed by gelatin zymograms. (D) Western blots analysis of MMP-9 protein levels. *, p<0.05 <i>vs</i> saline-treated brains (n = 4).</p

P188 improves long-term functional recovery after ischemia/reperfusion.

<p>(A) Post-stroke survival rate three weeks after stroke. *P<0.05, analyzed using log-rank test. (B) Wire hanging test. Time to stay hanging on the wire (T/hang). (C and D) Pole test. Time to turn the head downwards (T/turn). Time to reach the floor (T/floor). ^#, p<0.05 <i>vs.</i> sham-operated group; *, P<0.05 <i>vs.</i> saline-treated group. (E-G) Brain atrophy 3 weeks after tMCAO. Brain atrophy was determined using cresyl violet staining. The loss of brain volume was calculated as percentage of brain loss over total brain area of sham-operated mice.</p

Nutrient deprivation decreased cell viability when Bcl-2 was down-regulated.

<p>A: Western blot analysis of Bcl-2 expression in SH-SY5Y cells after transfection with negative control and Bcl-2 siRNA oligonucleotides. B: Quantitative analysis of optical densities of Bcl-2 protein bands (NC, negative control; Bcl-2 RNAi #6 and Bcl-2 RNAi #7) and normalized to the loading control. Bars represent Mean ± SD (n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. **p<0.01 represent Bcl-2 RNAi groups (#6 and #7) vs negative control group. C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells after transfection with NC, #6 and 7# oligonucleotides. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. ***p<0.001 represent the indicated groups vs NC starvation 12 hrs group; ###p<0.001 represent the indicated groups vs NC starvation 24 hrs group. D: Quantitative analysis of cell viability with MTT assay in SH-SY5Y control cells and the cells treated with DMSO, HA 14-1 and ABT-737. After pre-treated with HA 14-1 or ABT-737, cells were maintained either in normal medium or starvation medium containing DMSO, HA14-1 and ABT-737 for 12 hrs or 24 hrs prior to cell viability assay. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. ***p<0.001 represent HA 14-1/ABT-737 starvation 12 hrs groups vs DMSO starvation 12 hrs group; ###p<0.001 represent HA 14-1/ABT-737 starvation 24 hrs groups vs DMSO starvation 24 hrs group. For C and D, data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.</p

Knockdown of Beclin1 rescued Bcl-2 knockdown-induced cell death in response to starvation.

<p>A and B: Western blot analysis of Beclin1 expression in SH-SY5Y cells 72 hr after transfection and quantitative analysis of optical densities of Beclin1 protein bands with Sigma Scan Pro 5 and normalized to the loading control (B). Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). C and D: After Beclin1 was knocked down, the cells were subjected to serum starvation for another 12 hrs, and then Beclin1 and LC3 were detected (C) and quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5 (D, **p<0.01). Bars represent Mean ± SD (n = 3). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were co-transfected with Bcl-2, Beclin1 or Bcl-2/Beclin1 siRNA for 72 hrs, and the cells were subjected to serum starvation for 12 hrs or 24 hrs in the presence or absence of 50 µM Z-VAD. ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ###p<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.</p

Serum deprivation induced cell death when Bcl-2 function was suppressed.

<p>A: Flow cytometry analysis of SH-SY5Y control cells transfected with negative control, Bcl-2 RNAi #6 and Bcl-2 RNAi #7. The cells were either growing in normal medium or subjected to 12 hrs starvation. B: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried with Student t-test or carried out with ANOVA followed by Dunnett t-test (##p<0.01, ###p<0.001, Bcl-2 RNAi #6 + starvation, or #7 + starvation group vs NC+starvation group). C: Nutrient deprivation induced activation of caspase-3 (p19 and p17) and PARP cleavage in SH-SY5Y cells when Bcl-2 was knocked down. The results are representative of three experiments. D: Flow cytometry analysis of SH-SY5Y.control cells treated with DMSO, HA 14-1 and ABT-737. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining in SH-SY5Y cells treated with DMSO, HA 14-1 and ABT-737. Cells were either maintained in normal medium or subjected to 12 hrs of starvation prior to cell death analysis. Statistical analysis was either carried out with Student t-test or carried out with ANOVA followed by Dunnett t-test (###p<0.001, HA 14-1 + starvation, or ABT-737 + starvation group vs DMSO + starvation group). For B and E, data represent mean ± SD for combined data from three independent experiments.</p

Autophagic flux induced by serum deprivation was enhanced when Bcl-2 was down-regulated and inhibitors of autophagy and apoptosis rescue cell from death.

<p>A: Western blot analysis of LC3-II and p62 expression in SH-SY5Y cells. Seventy-two hours after transfection with indicated oligonucleotides, the cells were subjected to starvation for 12 hrs in the present or absence of 50 nM Baf A1. B: Quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). Statistical analysis was carried out with unpaired t-test (*p<0.05, Bcl-2 siRNA #6 group vs negative control group). C: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were treated with Bcl-2 siRNA #6 for 72 hrs, and then were treated with 10 µg/ml E64D, 50 nM Baf A1 and 50 µM Z-VAD. Cells were either maintained in normal medium or subjected to 12 hrs or 24 hrs of starvation prior to cell viability assay. **p<0.01, ***P<0.001 represent the indicated groups vs Bcl-2 RNAi #6 starvation 12 hrs group; ##p<0.01 represent the indicated groups vs Bcl-2 RNAi #6 starvation 24 hrs group. D: Representative western blot image of LC3 in SH-SY5Y cells subjected to normal or serum-free medium in the presence or absence of 10 µg/ml E64D for 12 hrs. E and F: Quantitative analysis of cell viability with MTT assay. SH-SY5Y cells were pre-treated with HA 14-1 or ABT-737, then the cells were either maintained in normal or starvation medium for 12 hrs or 24 hrs with E64D, Baf A1 or Z-VAD. **p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 12 hrs groups; #p<0.05, ##p<0.01 represent the indicated groups vs HA 14-1/ABT-737 starvation 24 hrs groups. Statistical analysis was either carried out with Student t-test, or carried out with ANOVA followed by Dunnett t-test. Data represent mean ± SD for combined data from three independent experiments, each experiment has six replicate wells.</p

Overexpression of Bcl-2 inhibited autophagy and blocked cell death.

<p>A: SH-SY5Y cells were transiently transfected with pCDNA3.1-Bcl-2 or empty control vector, and the cells were subjected to serum starvation for 12 hrs after 48 hrs transfection. B: Quantitative analysis of optical densities of the LC3-II/LC3-I protein bands with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). C: Quantitative analysis of cell viability with MTT assay in SH-SY5Y cells 24 hrs after transfection with control vector or Bcl-2 plasmid. Cells were either maintained in normal or starvation medium for 12 hrs prior to cell viability assay. D: Flow cytometry analysis of SH-SY5Y cells transfected with mock control and Bcl-2 plasmid for 48 hrs, then the cells were growing in normal medium or subjected to 24 hrs starvation. E: Quantitative analysis of cell death by flow cytometry Annexin V/PI staining. Statistical analysis was carried out with ANOVA followed by Dunnett t-test (*p<0.05). Data represent mean ± SD for combined data from three independent experiments. For MTT assay, each experiment has six replicate wells.</p

Autophagy was induced in SH-SY5Y neuroblastoma cells by serum deprivation.

<p>A: Representative confocal images (5 µM scale bar) of GFP-LC3 in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty-four hours after transfection, cells were treated with serum-free medium in the presence or absence of 100 nM Baf A1 for the indicated times. B: Quantification of autophagy in serum-starved SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD for combined data from three independent experiments. C: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium in the presence or absence of 100 nM Baf A1. E: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium. Each time course has a control. D and F: Quantitative analysis of optical densities of LC3-II/LC3-I with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). G and H: Western blot analysis (G) and quantitative analysis (H) of Beclin1 expression in SH-SY5Y cells subjected to serum-free medium. Bars represent Mean ± SD (n = 5). I and J: Western blot analysis (I) and quantitative analysis (J) of p62 expression in SH-SY5Y cells subjected to serum-free medium with and without NH₄Cl. Bars represent Mean ± SD (n = 3). K and L: Western blot analysis (K) and quantitative analysis (L) of cathepsin D expression in SH-SY5Y cells subjected to starvation. Bars represent Mean ± SD (n = 5). The optical densities of each protein band were quantified with Sigma Scan Pro 5 and normalized to the loading control. Statistical analysis was carried out with ANOVA followed by Dunnett t-test. (B and D) *p<0.05 and **p<0.01 vs control group. (I) #p<0.05 represent starvation vs starvation+NH₄Cl.</p