Hypoxia induces caspase3 expression without affecting cell viability in microglia.

A. Bar graph shows the viability of BV-2 cells after hypoxia for 12 h compared to control by MTS. Note no significant difference was observed in the two groups. B. PI- Annexin V staining determined by FACS analysis in microglia. C. Western blotting of caspase3 protein expression in BV-2 cells exposed to hypoxia for 2, 4, 6, 8 and 12 h and control (c). The left panel in C shows specific bands of cleaved caspase3 (17 kDa) and β-actin (43 kDa). The right panel in C is a bar graph showing significant changes in the optical density following hypoxic exposure (normalized with β-actin). Note significant increase in cleaved caspase3 expression after hypoxic treatment of varying durations in BV-2 cells, especially at 8 h. D. Confocal images showing cleaved caspase3 expression in control BV-2 cells and those exposed to hypoxia for 8 h. Weak cleaved caspase3 expression is detected in the control BV-2 cells with enhanced immunofluorescence intensity after 8 h of hypoxic exposure. The experiments have been repeated at least in triplicate. Significant differences between control and hypoxic BV-2 cells are calculated using Student’s t-test and expressed as *p .05 and **p .01. The values represent the mean ± SD in triplicate. Scale bar in D = 20 μm.</p

Caspase3 inhibitor reduces release of proinflammatory mediators and NF-κB activation.

Western blotting of TNF-α, IL-1β, iNOS and NF-κB protein expression in BV-2 cells following hypoxic exposure and Z-DEVD-FMK pretreatment. The upper panel shows specific bands of TNF-α (25.6 k Da), IL-1β (17k Da), iNOS (130 kDa), NF-κB /P65 (65k Da) and β-actin (43 kDa. The lower panels are bar graphs showing significant changes in the optical density in protein expression of different groups (normalized with β-actin). Note the decrease in TNF-α, IL-1β, iNOS and NF-κB expression in hypoxia+ Z-DEVD-FMK group compared with hypoxic BV-2 cells. The experiments have been repeated at least in triplicate. The statistical significance of differences between different groups was calculated using ANOVA. Significant difference between control vs hypoxia groups is shown as *pppp<0.01. The values represent the mean ± SD in triplicate.</p

Melatonin inhibits TLR4 mRNA and protein expression and caspase-3 activity in hypoxic BV-2 cells.

<p>A. Hypoxia induces TLR4 mRNA expression in hypoxic microglia that is suppressed by melatonin. B. BV-2 cells subjected to hypoxia, followed by western blot analysis of TLR4 and cleaved caspase-3 with or without melatonin treatment. The panel shows specific bands of TLR4 (95 kDa), cleaved caspase-3 (17 kDa) and β-actin (43 kDa). Note TLR4 and cleaved caspase-3 expression in melatonin-treated group is significantly decreased in comparison with the untreated group (normalized with β-actin). The experiments have been repeated at least in triplicate. The statistical significance of differences between different groups was calculated using ANOVA. Significant difference between control vs hypoxia groups is shown as *<i>p</i><0.05 and **<i>p</i><0.01; significant difference between hypoxia vs hypoxia +Melatonin groups is shown as #<i>p</i><0.05 and ##<i>p</i><0.01. The values represent the mean ± SD in triplicate.</p

Caspase3 activation is dependent on TLR4 expression in hypoxic microglia.

<p>Western blot analysis of caspase3 activation in control BV-2 cells, BV-2 cells + hypoxia, BV-2 cells transfected with control siRNA, BV-2 cells transfected with control siRNA + hypoxia, BV-2 cells transfected with TLR4 siRNA and BV-2 cells transfected with TLR4 siRNA + hypoxia. The upper panel shows specific bands of cleaved caspase3 (17 kDa) in different groups of BV-2 cells given different treatments. The lower panel are bar graphs showing significant changes in the optical density following hypoxic exposure (normalized with β-actin). The experiments have been repeated at least in triplicate. The statistical significance of differences between different groups was calculated using ANOVA. Significant difference between different groups is shown as *<i>p</i><0.05 and **<i>p</i><0.01. The values represent the mean ± SD in triplicate.</p

Melatonin suppresses the increase in TLR4 immunofluorescence in microglia in neonatal rats after hypoxic treatment.

<p>Confocal images showing TLR4 expression in lectin labeled (green) microglia in the corpus callosum of control, hypoxia and hypoxia+melatonin rats at 3 days after the hypoxic exposure. Increase in TLR4 expression in microglia is evident in hypoxic rats. Note TLR4 immunofluorescence intensity is attenuated in hypoxia+melatonin rats compared with that in the hypoxic rats. The lower graph showing the optical density of TLR4 on microglia of different groups. Note the optical density of TLR4 on microglia was increased in rats following hypoxia challenge and the optical density was decreased in hypoxia+melatonin group compared with hypoxia group. The experiments have been repeated at least in triplicate. The statistical significance of differences between different groups was calculated using ANOVA. Significant difference between control vs hypoxia groups is shown as *<i>p</i><0.05 and **<i>p</i><0.01; significant difference between hypoxia vs hypoxia+melatonin groups is shown as #<i>p</i><0.05 and ##<i>p</i><0.01. The values represent the mean ± SD in triplicate. Scale bar = 20 μm.</p

Primer sequences used for RT–PCR.

<p>Primer sequences used for RT–PCR.</p

LAT2 expression in AMC and BV2 microglia.

<p><b>A–F.</b> Confocal images showing the expression of LAT2 (B,E; red) in OX42-positive (A,D; green) microglia (C,F; yellow) from the corpus callosum of 5 &7days rat brains, respectively. <b>G–I.</b> Confocal images showing the expression of LAT2 (H; red) in BV2 microglial cells counter stained with lectin (G; green) and DAPI (blue).</p

Cdk4 mRNA and protein expression in BV2 microglia treated with HDACi.

<p><b>A.</b> qRT-PCR analysis shows that Cdk4 mRNA expression is significantly upregulated in LPS-treated BV2 microglia and however, downregulated when cells are treated with HDACi. <b>B–C.</b> Western blot analysis also showed a similar trend ofCdk4 (34 kDa) expression in BV2 microglia treated with HDACi or LPS or both. The data are normalized with β-actin (42 kDa) immunoreactivity (B). Significant differences between groups (control <i>vs</i> LPS, LPS <i>vs</i> HDACi and LPS <i>vs</i> HDACi +LPS) are indicated by *<i>p</i><0.05, **<i>p</i><0.01 (n = 3).</p

Confocal images showing the BrdUimmunostaining in lectin-positive BV2 microglia (A,C,E,G; red).

<p>The number of BrdUpositive microglial cells (C,G: arrows) appears to be decreased at 24 h after Runx1t1 knockdown when compared to the cells transfected with scrambled siRNA. Nuclei are stained with DAPI. Scale bars: 50 µm. <b>I.</b> Quantitative analysis shows a significant decrease in number of BrdUimmunoreactive cells in activated microglia cultures transfected with Runx1t1siRNA for 24 h. Significant differences between groups (scrambled siRNA <i>vs</i> Runx1t1 siRNA, scrambled siRNA +LPS <i>vs</i> Runx1t1 siRNA+LPS) are indicated by *<i>p</i><0.05, **<i>p</i><0.01 (n = 3).</p

Cdk4 protein expression in LPS-activated Runx1t1 knockdown BV2 microglia.

<p><b>A–B.</b> Western blot analysis shows that Cdk4 protein expression is upregulated in LPS-treated BV2 microglial cells (A–B). However, a significant downregulation of Cdk4 protein expression is detected in LPS-treated BV2 microglia after knockdown of Runx1t1 (A–B). The significant difference between the groups (scrambled siRNA <i>vs</i> scrambled siRNA +LPS, Runx1t1 siRNA <i>vs</i> Runx1t1 siRNA+LPS and scrambled siRNA +LPS <i>vs</i> Runx1t1 siRNA+LPS) are indicated by *<i>p</i><0.05, **<i>p</i><0.01(n = 3).</p