Fig. 5

Autophagic modulation changes Hcy-induced proteotoxicity and consequent neuronal cell death. A Representative immunoblot showing autophagic blocker BafA1 (20 nM) has equal or additive effect on polyubiquitinated protein load in Hcy alone or combined treatment in SK-N-SH cells. β-actin was used as a loading control. B Bars showing increased percent cell death by BafA1 in Hcy-treated neuronal cells. C Representative confocal images confirming increased mitochondrial fragmentation by BafA1 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 population. Scale bar 10 µm. Pharmacological activation of autophagy by RAPA (200 nM) showing rescue of polyubiquitinated protein load in immunoblots (D) and consequent protection against percent cell death (E) in neuronal cells upon Hcy treatment. β-actin was used as a loading control in immunoblot analysis. F Representative confocal images showing the rescue of mitochondrial fragmentation by RAPA in Hcy treated primary neurons. Insets showing zoomed portions of the images and bar graph showing the average length distribution of different mitochondrial populations. Scale bar 10 µm. Genetic activation of autophagy showing rescue of percent cell death (G) and ubiquitinated protein load (H) upon Hcy treatment in B35 neuroblastoma cells stably overexpressing ATG7. GAPDH was used as a loading control in immunoblot analysis. Concentration of Hcy was 5 mM for SK-N-SH cells and 0.75 mM for primary neurons. Data are shown as mean ± SEM with n ≥ 3. **P < 0.01. ****P < 0.0001