Figure 5

Vitamin B₁₂ and folate prevented Hcy-induced decrease in lysosomal pathway. (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 key lysosomal proteins was done and plotted as bar graphs. (e) SH-SY5Y neuroclastoma cells were treated with Hcy (2.0 mM for 48 h) alone or along with Vitamin B₁₂ and folate (1 : 1; 2 mM); and stained with AO for 30 min. Acidic compartments (lysosomal vesicles as RED) and nucleus (as GREEN) were observed under fluorescence microscope and captured using LAS software, analyzing red puncta formation under confocal microscope at 20 × magnification. Representative micrographs are shown here and enlarged view in the inset. (f) Relative fluorescence of lysosomal vesicles (RED) and nucleus (GREEN) was measured using ImageJ software (NIH, USA) and plotted as bar graph. The data are shown as Mean±S.D. and statistical difference *P<0.05 was considered as significant. (g and i) 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). (h and j) Quantitative analysis of key lysosomal proteins was done and plotted as bar graphs. (k) Primary human astrocytes treated with Hcy (2.0 mM for 48 h) alone or along with Vitamin B₁₂ and folate (1 : 1; 2 mM), RT-qPCR analysis of key lysosmal, autophagy and ER stress pathway genes was performed and plotted as bar graph. The data are shown as Mean±S.D. and statistical difference *P<0.05 was considered as significant