Fig. 2: LATS1 restricts autophagy in a kinase activity-independent manner.
From: LATS1 but not LATS2 represses autophagy by a kinase-independent scaffold function
a, b Immunofluorescence analysis of LC3B puncta in Huh7 cells in response to sorafenib (Srf) treatment. Cells transfected with indicated siRNAs were treated with DMSO or sorafenib (6 μM for Huh7) for 40 h and bafilomycin A1 (Baf; 0.1 μM) for 2 h. Representative images (a) and quantification of LC3B puncta numbers (b) from three independent experiments are shown. Statistical analysis was calculated by one-way ANOVA. Scale bars, 25 μm. c The forced expression of both wild-type (WT) LATS1 and a kinase-dead (KD D846A) version of LATS1 blocks sorafenib-induced autophagic flux in Hep3B cells. Results represent three independent experiments. d, e Huh7 cells transfected with either siControl or siRNA against LATS1 were in addition transfected with empty vector (EV) or a vector encoding siRNA-refractory wild-type (WT) or a kinase-dead (KD D846A) version of LATS1, as indicated. Representative immunoblots (d) and quantification of the relative p62 levels (e) (normalized to GAPDH, fold change) from three independent experiments are shown. Statistical significance was calculated using one-way ANOVA. f Flow cytometry analysis of autophagy induction in U2OS-GFP-LC3 cells. Cells were transfected with indicated siRNAs and then treated with rapamycin (100 nM) for 16 h and additional chloroquine (10 μM) for 2 h. Results represent for three independent experiments. g, h Control mice (Lats1fl/fl; Control) or mice with a hepatocyte-specific depletion of Lats1 (Alb-Cre;Lats1fl/fl; cKO) were treated with chloroquine (30 mg/kg) for 4 h. Liver samples were collected and lysed for immunoblotting analysis. An immunoblot (g) and quantification of the relative LC3BII levels (h) (normalized to GAPDH, fold change) from n = 3 mice per cohort are shown. Statistical significance was calculated using two tailed, unpaired t-test. i, j Control mice (Lats1fl/fl; Control) or mice with a hepatocyte-specific depletion of Lats1 (Alb-Cre;Lats1fl/fl; cKO) were treated with rapamycin (2 mg/kg, twice within 24 h) followed with being fasted for 12 h. Liver samples were collected and lysed for immunoblotting analysis. An immunoblot (i) and quantification of the relative p62 levels (j) (normalized to GAPDH, fold change) in livers of n = 4 for control and n = 6 for cKO mice. Statistical significance was calculated using two-tailed unpaired t-test.
