Figure 8. Protrudin depletion promotes initiation of autophagy. (A) Immunoblot showing an increased level of LC3-II in fed RPE-1 cells transfected with siRNA targeting Protrudin compared with control. The graph represents the relative level of LC3-II normalized to the loading control HSP90 quantified from immunoblots as in A. Error bars denote ± SEM from independent experiments (n = 5). *, P < 0.02; **, P < 0.01 (one-sample t test). (B) RPE-1 cells were siRNA transfected and either grown in complete medium or starved for 2 h in EBSS. ConA was given for 2 h where indicated. The cells were stained with an antibody to LC3 and analyzed by high-content wide-field microscopy. (C) Automated quantification of images as in B of the total intensity of LC3 dots per cell using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 4). ***, P < 0.001 (unpaired t test). More than 1,400 cells were analyzed per condition. (D) Automated quantification of images as in B of the number of LC3 dots per cell using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 4). **, P < 0.01; ***, P < 0.001 (unpaired t test). More than 1,400 cells were analyzed per condition. (E) Automated quantification of images as in B of the amount of cells having LC3 dots with size >50 pixels, using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 4). **, P < 0.01; ***, P < 0.001 (unpaired t test). More than 1,400 cells were analyzed per condition. (F) Imaris representations of confocal z-stacks visualizing the number and size of LC3 positive dots in control- or siRNA-treated RPE-1 cells grown in complete medium with or without ConA. (G) Quantification of the rate of LLPD in control and siRNA-treated RPE-1 cells. Error bars denote ± SEM from independent experiments (n = 3). ***, P < 0.001 (unpaired t test).
Figure 8.

Protrudin depletion promotes initiation of autophagy. (A) Immunoblot showing an increased level of LC3-II in fed RPE-1 cells transfected with siRNA targeting Protrudin compared with control. The graph represents the relative level of LC3-II normalized to the loading control HSP90 quantified from immunoblots as in A. Error bars denote ± SEM from independent experiments (n = 5). *, P < 0.02; **, P < 0.01 (one-sample t test). (B) RPE-1 cells were siRNA transfected and either grown in complete medium or starved for 2 h in EBSS. ConA was given for 2 h where indicated. The cells were stained with an antibody to LC3 and analyzed by high-content wide-field microscopy. (C) Automated quantification of images as in B of the total intensity of LC3 dots per cell using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 4). ***, P < 0.001 (unpaired t test). More than 1,400 cells were analyzed per condition. (D) Automated quantification of images as in B of the number of LC3 dots per cell using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 4). **, P < 0.01; ***, P < 0.001 (unpaired t test). More than 1,400 cells were analyzed per condition. (E) Automated quantification of images as in B of the amount of cells having LC3 dots with size >50 pixels, using the Olympus ScanR system. Error bars denote ± SEM from independent experiments (n = 4). **, P < 0.01; ***, P < 0.001 (unpaired t test). More than 1,400 cells were analyzed per condition. (F) Imaris representations of confocal z-stacks visualizing the number and size of LC3 positive dots in control- or siRNA-treated RPE-1 cells grown in complete medium with or without ConA. (G) Quantification of the rate of LLPD in control and siRNA-treated RPE-1 cells. Error bars denote ± SEM from independent experiments (n = 3). ***, P < 0.001 (unpaired t test).

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