Figure 2.

Baclofen protected RGCs against CoCl₂-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 CoCl₂ 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 CoCl₂ 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 CoCl₂ 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 CoCl₂ 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.