Fig. 2

ER stress generated by Hcy causes mitochondrial dysfunction and neuronal toxicity. A Representative immunoblots showing expression levels of ER stress markers in Hcy treated SK-N-SH (left) and primary neurons (right). β-actin was used as a loading control. Densitometric values in the blots represent the ratio of respective protein signal to β-actin signal normalized to control. B Bar graphs showing rescue of Hcy-induced cell death in SK-N-SH and primary neurons by the chemical chaperone; 4-PBA. C Immunoblot analysis showing that only Hcy-condition causes caspase-3 activation (Cle-Casp-3) from the inactive pool of procaspase-3 (Pro-Casp-3) and the blockage in cleavage and activation of caspase-3 by 4-PBA in Hcy-treated primary neurons. Arrows marking the bands for respective inactive and active forms of Caspase-3. D Representative confocal images showing the rescue of mitochondrial fragmentation by 4-PBA in mitoDsRed expressing SK-N-SH cells upon Hcy treatment. Insets showing zoomed portions of the images and bar graph showing the average length distribution of different mitochondrial populations. Scale bar 20 µm. E Representative immunoblot showing time dependent increase in polyubiquitinated protein load in Hcy-treated primary neurons. GAPDH was used as a loading control. F Visualization of the increase in polyubiquitinated protein puncta by immunocytochemistry in Hcy-treated SK-N-SH cells (upper panel). Confocal images with scale bar 20 µm. Bar diagram (lower panel) showing the percent count of puncta containing cells. G Representative immunoblot showing the rescue of polyubiquitinated protein load by 4-PBA in Hcy treated SK-N-SH cells. β-actin was used as a loading control. Concentration of Hcy was 5 mM for SK-N-SH cells and 0.75 mM for primary neurons. 1 mM of 4-PBA was used. Data are shown as mean ± SEM with n ≥ 3. **P < 0.01.***P < 0.001.****P < 0.0001