Extended Data Fig. 8: Rab24 KD caused an increase in p62 independent autophagic flux during fasting. | Nature Metabolism

Extended Data Fig. 8: Rab24 KD caused an increase in p62 independent autophagic flux during fasting.

From: Hepatic Rab24 controls blood glucose homeostasis via improving mitochondrial plasticity

Extended Data Fig. 8

Quantification of LC3-II/VCP levels in primary hepatocytes (a) and liver tissue (b) in fed, starved or chloroquine treated conditions (mean +/- SD). Cells were kept in full medium (fed) or serum starved for 12 h (fasted), followed by 20 µM chloroquine treatment for 3 h (fasted & CQ). Mice were either fed ad libitum, fasted for 12 h, or fasted for 12 hours followed by 100 mg/kg chloroquine treatment for 3 h (fasted & CQ). (c) Steady state levels (fed condition) of LC3-II staining in primary hepatocytes (mean +/- SEM, N=14 for CTR and N=12 for KD cellular regions (multiple cells per region) analyzed). (d) Representative Western blots and quantification thereof for p62 in primary hepatocytes (e) and liver tissue (f) in fed, starved or chloroquine treated conditions (mean +/- SD). The quantifications in (a) and (e) are from six independent wells of a 24-well plate pooled into N=2 replicates. The quantifications in (b) and (f) and the Western blot in (d) are from N=2 animals per condition. Both experiments were done twice with similar results. (g) Representative confocal images (three merged confocal section) of primary hepatocytes stained with dapi and p62 (grey) and quantification thereof with Fiji in (h) after treatment with full medium (fed) or serum starved for 12 h (fasted), followed by 20 µM chloroquine treatment for 3 h (fasted & CQ). The images are representative of N=12 (fed), N=20 (starved), N=12 (starved & CQ) for CTR and N=8 (fed), N=20 (starved), N=12 (starved & CQ) cellular regions (multiple cells per region) analyzed. (i) Representative confocal images (single middle confocal sections) of primary polarized hepatocytes in collagen sandwich stained for LAMP1 (grey), phalloidin (green), and dapi (blue) and quantification of the mean fluorescent intensity of LAMP1 per cell using Fiji in j. Scale bar = 20 μm. The images are representative of three independent wells of a 24-well plate. N=40 (CTR) and N=29 (KD) cells per condition. The in vitro experiment was repeated twice with similar results. All measured after 3 days of RNAi against Rab24 (40 nM) in primary hepatocytes (mean +/- SEM). *P<0.05, ****P<0.0001 by two-tailed unpaired Student’s t-test.

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