Figure 5.
Depletion of migfilin does not lead to lysosome dysfunction. (A) Representative images showed that the number of lysosomes labeled by mCherry-Lamp1 in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of mCherry-Lamp1 labeled lysosomes was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 27 cells for each group. (B) Representative images showed that the number of functional lysosomes indicated by lysotracker red (Lyso-red) in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 5 µm. Quantification of the number of functional lysosomes labeled by lysotracker red was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 54 cells for each group. (C) Flow cytometry analysis of Lyso-Tracker Red positive cells in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. (D) Representative images showed that lysosomal acidities indicated by lysosensor green (LysoSen Green) in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 2 µm. Quantification of the lysosomal acidities was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 12 images containing >30 cells for each group. (E) Flow cytometry analysis of Lyso-Sensor Green positive cells in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. (F) Representative images showed that lysosomal activities (Cathepsin L activity) measured by Magic Red Cathepsin L kit in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 2 µm. Quantification of the lysosomal activities was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 21 cells for each group. (G) Representative images showed that lysosomal digestive activities measured by DQ-BSA in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 5 µm. Quantification analysis was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 13 images containing >30 cells for each group. (H) Flow cytometry analysis of DQ-BSA positive cells in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition.

Depletion of migfilin does not lead to lysosome dysfunction. (A) Representative images showed that the number of lysosomes labeled by mCherry-Lamp1 in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 10 µm (Magnify, 2 µm). Quantification of the number of mCherry-Lamp1 labeled lysosomes was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 27 cells for each group. (B) Representative images showed that the number of functional lysosomes indicated by lysotracker red (Lyso-red) in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 5 µm. Quantification of the number of functional lysosomes labeled by lysotracker red was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 54 cells for each group. (C) Flow cytometry analysis of Lyso-Tracker Red positive cells in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. (D) Representative images showed that lysosomal acidities indicated by lysosensor green (LysoSen Green) in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 2 µm. Quantification of the lysosomal acidities was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 12 images containing >30 cells for each group. (E) Flow cytometry analysis of Lyso-Sensor Green positive cells in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. (F) Representative images showed that lysosomal activities (Cathepsin L activity) measured by Magic Red Cathepsin L kit in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 2 µm. Quantification of the lysosomal activities was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 21 cells for each group. (G) Representative images showed that lysosomal digestive activities measured by DQ-BSA in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition. Scale bar, 5 µm. Quantification analysis was shown in the right panel. Data represent mean ± SE, n.s., no significance, unpaired two-tailed t test. n = 3 independent experiments with at least 13 images containing >30 cells for each group. (H) Flow cytometry analysis of DQ-BSA positive cells in control (siCtrl) and migfilin knockdown (siMig #1) KP4 cells under control or starvation condition.

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