Journal: Frontiers in Cellular Neuroscience

Article Title: Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways

doi: 10.3389/fncel.2016.00255

Figure Lengend Snippet: Baclofen protected RGCs against CoCl 2 -induced apoptosis without interfering cell viability of RGCs. (A) Cell viability detected by CCK-8 assay in RGCs treated with indicated concentrations of baclofen for 24 h. Data are means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 vs. basal level. (B,C) Expression of cleaved caspase-3, bax and bcl-2 detected by Western blotting in RGCs treated with indicated concentrations of CoCl 2 and baclofen for 24 h. The detected protein bands are normalized to the signal of α-tubulin. The protein levels were expressed as the value relative to the control. The data represent the mean ± SD of three independent experiments. (* p < 0.05, ** p < 0.01 compared to control). (D,E) Cell apoptosis detected by Annexin V and PI staining methods in RGCs treated with indicated concentrations of CoCl 2 and baclofen for 24 h. C4 quadrant (Annexin V + /PI−) indicates the percentage of apoptotic cells. Values represent the mean ± SD of three independent experiments. ** P < 0.01 vs. basal level. (F,G) RGCs stained with Hoechst nuclear stain after treatment with indicate concentrations of CoCl 2 and baclofen for 24 h, and representative experiments are shown. Values represent the mean ± SD of three independent experiments. * P < 0.05 vs. basal level. White arrows indicate the apoptotic nuclei. (H,I) Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to evaluate hypoxia-induced cell death. Cells were exposed to indicate concentrations of CoCl 2 and baclofen for 24 h. Apoptotic nuclei were visualized by TUNEL. Representative experiments are shown (Final magnification, ×40). Values represent the mean ± SD of three independent experiments. ** P < 0.01 vs. basal level. White arrows indicate the apoptotic nuclei. (J,K) TUNEL-positive cells detected by flow cytometry. Representative experiments are shown. Values represent the mean ± SD of three independent experiments. *** P < 0.001 vs. basal level.

Article Snippet: After being fixed with 4% paraformaldehyde at 4°C for 30 min, cells were incubated with the DNA stain Hoechst 33,342 (Beyotime, Shanghai, China) for 10 min at room temperature and mounted with a fluorescent mounting medium (Beyotime, Shanghai, China).

Techniques: CCK-8 Assay, Expressing, Western Blot, Control, Staining, End Labeling, TUNEL Assay, Flow Cytometry

Journal: Frontiers in Cellular Neuroscience

Article Title: Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways

doi: 10.3389/fncel.2016.00255

Figure Lengend Snippet: Baclofen mediated RGCs apoptosis via GABA B receptor. (A,B) RGCs were transfected with GABA B 2 siRNA2 or control siRNA before treatment with CoCl 2 and baclofen for 24 h as indicated. Cell apoptosis detected by Annexin V and PI staining methods. C2, C4 quadrant (Annexin V + /PI−) indicate the percentage of necrotic or apoptotic cells, respectively. Values represent the mean ± SD of three independent experiments. ** P < 0.01 vs. basal level. (C,D) RGCs stained with Hoechst nuclear stain after treatment as described above. White arrows indicate the apoptotic nuclei. Values represent the mean ± SD of three independent experiments. * P < 0.05 vs. basal level. (E–H) RGCs were transfected with GABA B 2 siRNA2 or control siRNA before treatment with CoCl 2 and baclofen. Expression of apoptosis related proteins (E,F) and pathway related proteins (G,H) detected by Western blotting treated with baclofen and CoCl 2 for 24 h as presented. α-tubulin served as a loading control. The level of protein in each group was expressed as the value relative to the control. The data represent the mean ± SD of three independent experiments. (* p < 0.05, vs. control).

Article Snippet: After being fixed with 4% paraformaldehyde at 4°C for 30 min, cells were incubated with the DNA stain Hoechst 33,342 (Beyotime, Shanghai, China) for 10 min at room temperature and mounted with a fluorescent mounting medium (Beyotime, Shanghai, China).

Techniques: Transfection, Control, Staining, Expressing, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Baclofen Protects Primary Rat Retinal Ganglion Cells from Chemical Hypoxia-Induced Apoptosis Through the Akt and PERK Pathways

doi: 10.3389/fncel.2016.00255

Figure Lengend Snippet: Akt phosphorylation is needed for baclofen mediating RGCs apoptosis. (A,B) Cell apoptosis detected by Annexin V and PI staining methods in RGCs treated with CoCl 2 , baclofen and Akt inhibitor for 24 h as indicated. C4 quadrant (Annexin V + /PI−) indicates the percentage of apoptotic cells. Values represent the mean ± SD of three independent experiments. ** P < 0.01 vs. basal level. (C,D) RGCs stained with Hoechst nuclear stain after treatment as described above. White arrows indicate the apoptotic nuclei. Values represent the mean ± SD of three independent experiments. ** P < 0.01 vs. basal level. (E–H) Expression of apoptosis related proteins (E,F) and pathway related proteins (G,H) detected by Western blotting in RGCs treated with baclofen, CoCl 2 and Akt inhibitor for 24 h as presented. α-tubulin was included as a loading control. Protein levels were expressed as the value relative to the control in each group. The data represent the mean ± SD of three independent experiments. (* p < 0.05, vs. control).

Article Snippet: After being fixed with 4% paraformaldehyde at 4°C for 30 min, cells were incubated with the DNA stain Hoechst 33,342 (Beyotime, Shanghai, China) for 10 min at room temperature and mounted with a fluorescent mounting medium (Beyotime, Shanghai, China).

Techniques: Phospho-proteomics, Staining, Expressing, Western Blot, Control