Figure 4

Vitamin B₁₂ and folate prevented Hcy-induced autophagic inhibition. (a and c) Western blot analysis of primary mouse and human astrocytes, respectively treated with Hcy (2.0 mM for 48 h) alone or along with Vitamin B₁₂ and folate (1 : 1; 2 mM). (b and d) Quantitative analysis of MAP1LC3B-II and SQSTM1 proteins was done and plotted as bar graphs. The data are shown as Mean±S.D. and statistical difference *P<0.05 was considered as significant. (e) SH-SY5Y neuroclastoma cells were transfected with ptf-LC3B and treated with Hcy (2.0 mM for 48 h) alone or along with Vitamin B₁₂ and folate (1 : 1; 2 mM). autophagic flux analysis was then performed by analyzing red puncta formation under confocal microscope at 40 × magnification. Representative micrographs are shown here and enlarged view in the inset. (f and h) Western blot analysis of primary mouse and human astrocytes, respectively, treated with Hcy (2.0 mM for 48 h) alone or along with Vitamin B₁₂ and folate (1 : 1; 2 mM). (g and i) Quantitative analysis of key MTOR signaling proteins was done and plotted as bar graphs. The data are shown as Mean±S.D. and statistical difference *P<0.05 was considered as significant