Lysosomal translocation of GAPDH-HT reflects CMA activity in HeLa cells.

<p>(<i>A</i>) Representative fluorescent images of GAPDH-HT 21 h after labeling with TMR-HT ligand in HeLa cells transfected with nontargeting-siRNA (left) or LAMP2A-siRNA (right). Bar = 20 µm. (<i>B,C</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in cells transfected with nontargeting (Non)- and LAMP2A-siRNA. (<i>B</i>) Cells having more than 5 dots of GAPDH-HT were classified as GAPDH-HT dot-positive cells. We counted the number of GAPDH-HT dot-positive cells in 50–70 GAPDH-HT-expressing cells. (<i>C</i>) We assessed the number of GAPDH-HT dots per cell. The percentage of GAPDH-HT dot-positive cells and the number of GADPH-HT dots per cell were significantly decreased by siRNA-mediated LAMP2A-knockdown. ** p<0.001 vs cells treated with nontargeting-siRNA (unpaired <i>t</i>-test, n = 16 in <i>B</i>, n = 57 for nontargeting-siRNA and n = 74 for LAMP2A-siRNA in <i>C</i>). (<i>D</i>) Representative fluorescent images of GAPDH-HT taken 21 h after labeling with the TMR-HT ligand in HeLa cells treated with vehicle (0.1% DMSO, 0.1% methanol, left upper), serum free medium (0.1% DMSO, 0.1% methanol, center upper), H₂O₂ (100 µM, right upper), mycophenolic acid (MPA; 10 µM, left lower), SB202190 (20 µM, left center) or cycloheximide (CHX; 20 µg/ml, right lower). Bar = 20 µm. (<i>E,F</i>) Quantitative analyses of GAPDH-HT lysosomal translocation in cells treated with CMA activators or inhibitors the percentages of GAPDH-HT dot-positive cells (<i>E</i>) and the number of GAPDH-HT dots per cell (<i>F</i>). Percentages of GAPDH-HT dot-positive cells and the numbers of GAPDH-HT dots per cell were significantly increased by CMA activators (serum free medium, H₂O₂ and MPA), while they were significantly decreased by CMA inhibitors (SB202190 and CHX). * p<0.01, ** p<0.001 vs cells treated with vehicle (unpaired <i>t</i>-test, n = 12 for cells treated with vehicle, n = 8 for cells treated with CMA activators and inhibitors in <i>E</i>, n = 96 for vehicle, n = 61 for serum free, n = 45 for H₂O₂, n = 58 for MPA, n = 52 for SM202190 and n = 41 for CHX in <i>F</i>).