Fig 2. TJ-35 suppresses autophagy under starvation condition.

A. Tf-LC3–expressing HeLa cells were treated with or without TJ35 for 4 h, shifted to DMEM or EBSS with or without TJ-35 for 2 h, and observed on SP-8. The graph above shows GFP-positive puncta per cell. Median: line; upper and lower quartiles: boxes; 1.5-interquartile range: whiskers. We counted 15 cells in three independent experiments. Bar represents 10 μm. The graph below shows the signal intensity ratio of GFP/RFP in each field of view after 6 h. B. HeLa cells were cultured in DMEM for 24 h, treated with or without TJ-35 for 4 h, and shifted to EBSS in the presence of TJ-35 for 2 h. The cells were immunostained with anti-LC3 antibody. The graph shows Alexa Fluor 488-positive puncta per cell. Median: line; upper and lower quartiles: boxes; 1.5-interquartile range: whiskers. We counted 25 cells in three independent experiments. Bar represents 10 μm. C. HeLa cells were treated with or without TJ35 in DMEM or EBSS, with or without bafilomycin A₁, for 4 h. The lysates were assessed by Western Blotting with LC3 antibody. The graph shows the average and standard deviation of LC3 signal versus tubulin signal from three independent experiments. * denotes p<0.05 (unpaired two-tailed Student’s t-test) between EBSS and EBSS plus TJ-35.