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. 2022 Jan 26;79(2):95. doi: 10.1007/s00018-022-04144-1

Fig. 6.

Fig. 6

KRT8 and PLEC is required for autolysosome formation in macroautophagy caused by MTOR inhibition condition. a Confocal microscopic images in mCherry-EGFP-LC3B-expressing ARPE-19 cells. Cells were incubated in the absence or presence of Torin1 for 24 h. Enlarged images of the white boxed areas are shown in the lower panels. Scale bar: 5 μm. Bar graph indicates the percentage of each fluorescence level in the merged images of mCherry-EGFP-LC3B under Torin1 treatment condition. Data are presented as the mean ± SEM, n = 3. ***P < 0.001. b Confocal microscopic images of Lysotracker Red-stained and GFP-LC3B-transfected ARPE-19 cells under Torin1 treatment for 24 h. Scale bar: 5 μm. Bar graph indicates the percentage of each fluorescence level in the merged images of GFP-LC3B and Lysotracker Red under the MTOR inhibition condition. Data are presented as the mean ± SEM, n = 3. ***P < 0.001. c Immunoblots of KRT8, SQSTM1, and LC3B in ARPE-19 cells treated with Torin1 for 24 h. Bar graphs indicate SQSTM1 expression level or the ratio of LC3B-II to LC3B-I. Each protein band intensity was normalized to GAPDH. Data are presented as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01. d Confocal microscopic images of mCherry-EGFP-LC3B-expressing ARPE-19 cells. Cells were transfected with anti-PLEC siRNA and incubated with Torin1 for 24 h. Scale bar: 5 μm. Bar graph indicates the percentage of each fluorescence level in the merged images of mCherry-EGFP-LC3B under Torin1 treatment condition. Data are presented as the mean ± SEM, n = 3. ***P < 0.001. e Confocal microscopic images of LysoTracker Red-stained and GFP-LC3B-transfected ARPE-19 cells. Cells were transfected with anti-PLEC siRNA and incubated with Torin1 for 24 h. Scale bar: 5 μm. Bar graph indicates the percentage of each fluorescence level in the merged images of GFP-LC3B and Lysotracker Red under the MTOR inhibition condition. Data are presented as the mean ± SEM, n = 3. ***P < 0.001. f Immunoblots of PLEC, SQSTM1, and LC3B in anti-PLEC siRNA-transfected ARPE-19 cells. Cells were treated with Torin1 for 24 h. Bar graphs indicate SQSTM1 expression level or the ratio of LC3B-II to LC3B-I. Each protein band intensity was normalized to GAPDH. Data are presented as the mean ± SEM, n = 3. *P < 0.05