Fig. 3

Elevated Hcyo downregulates basal autophagy in neuronal cells. A Left panel: representative confocal images showing LC3-GFP puncta in control and Hcy-treated SK-N-SH cells transiently transfected with LC3-GFP construct. Arrowheads represent GFP-LC3 puncta. Scale bar 20 µm. Right panel: percent quantitation of LC3-GFP puncta containing cells (≥ 10 puncta per cell). B Representative immunoblot (upper panel) and densitometric quantitation (lower panel) showing a time dependent decrease in lipidated LC3-II form (marker of autophagy) in Hcy-treated cells. β-tubulin was used as a loading control. C Representative TEM images showing the decrease in autophagic vesicles upon Hcy treatment in SK-N-SH cells. Arrowheads represent double membrane autophagic vesicles. Scale bar 0.5 µm. Bar graph showing the quantitation of the TEM images representing the number of autophagic vesicles per cell. D Representative immunoblots showing a decreased expression of major autophagic proteins (Beclin1, ATG5, ATG7) and a concomitant increase in cargo protein (P62) in SK-N-SH (left) and primary neurons (right), confirming autophagic suppression by Hcy. 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. 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 < 0.05. **P < 0.01