Fig 2: Depletion of CNOT2 induces autophagy via accumulation of p62/SQSTM1 and LC3B-II conversion, LC3 fluorescent puncta and autophagosomes, but impairs autophagic flux in H1299 cells(A) Western blotting was conducted for p62/SQSTM1 and LC3B-II in H1299 cells transfected with CNOT2 siRNA. (B) ImageJ densitometric analysis of the relative CNOT2, p62/SQSTM1 and LC3B-II protein expression levels (means ± SD of 3 independent experiments, *p < 0.05 vs untreated control by Student t test). (C) Accumulation of LC3 fluorescent puncta in H1299 cells transfected with CNOT2 siRNA compared to untreated control in H1299 cells (means ± SD of 3 independent experiments, * p < 0.05 vs untreated control by Student t test). (D) A number of autophagosomes and autophagolysosomes were observed in H1299 cells transfected with CNOT2 siRNA by TEM. The below panel is the enlarged image for the black frame. Autophagosomes and autophagolysosomes were represented by white and black arrowheads, respectively. Scale bar regular image: 10 μm, enlarge image: 2 μm. (E) Quantitative analysis of autohagosome and autophagolysosome in H1299 cells transfected with CNOT2 siRNA (means ± SD of 3 independent experiments, *** p < 0.001 vs untreated control by Student t test). (F) Effect of CNOT2 depletion on the expression of p62 and LC3B-II in CNOT2 depleted H1299 cells in a time course. (G) Depletion of CNOT2 inhibited autophagic flux in H1299 cells. Representative confocal images were exhibited in H1299 cells co-transfected with siCNOT2 and RFP-LC3 and GFP-LC3 constructs. Autophagosome was visualized as yellow or orange puncta (RFP-GFP-LC3B) in merged images, whereas red puncta (RFP-LC3B) represents autophagolysosome, since acidification reduces green fluorescence. Scale bar: 10 μm. (H) Quantitative analysis of LC3-puncta in H1299 cells transfected with CNOT2 siRNA in the present or absence of autophagy inhibitor 3-MA or NH4Cl (means ± SD of 3 independent experiments, * p < 0.05 vs untreated control by Student t test). (I) Effect of autophagy inhibitors on sub G1 population in CNOT2-depleted H1299 cells. Cells were transfected with CNOT2 siRNA for 24 h and treated with 3-MA, CQ and NH4Cl for another 24 h. Then the cells were fixed with 75 % ethanol and stained with 50 mg/ml of PI. The cell cycle analysis was performed using FACs Calibur, and the data were analyzed using CellQuest Software (means ± SD of 3 independent experiments, ** p < 0.01, *** p < 0.001 vs untreated control by Student t test). (J) Effect of autophagy inhibitors on PAPR cleavage and procaspase 8 in H1299 cells transfected with CNOT2 siRNA. Cells were transfected with CNOT2 siRNA for 48 h, exposed to 3MA or CQ for 24 h and Western blotting was conducted with antibodies of PAPR cleavage, procaspase 8, CNOT2 and actin.

Fig 3: Depletion of TAILS candidate proteins in CVB3-infected cells. Immunoblots of HeLa cells transfected with control siRNA (siSCR) or siRNAs directed against (A) AIMP2, (B) EMD, (C) CNOT2, or (D) RAI. siRNA-treated cells (48 h) were infected with CVB3 with an MOI 1.0 (A, B, C) or 0.1 (D) for 16 h. Extracellular and intracellular viral yields were detected by plaque assay. Shown are box plots of log10 PFU with means and 95% confidence intervals from three independent experiments with at least two technical replicates. (E) Extracellular and intracellular viral titers collected from CVB3-infected (MOI 0.1) HeLa cells were pre-treated with cerulenin (45 M) and ACA (20 M). P-values from nested one-way t-test are indicated.

Fig 4: CNOT2 increases ubiquitination and degradation of p62/SQSTM1 protein in HEK293 QBI cellsHEK293 QBI cells were co-transfected with Flag-p62 and HA-CNOT2 plasmids. Thereafter, the cells were cultured for 48 h and subjected to (A) western blot or (B) RT-PCR (means ± SD of 3 independent experiments, *** p < 0.001 vs control by Student t test). (C) Cells were transiently transfected with CNOT2 siRNA for 48 h and then subjected to immunoblotting with indicated antibodies (means ± SD of 3 independent experiments, ** p < 0.01 vs control by Student t test). (D) HEK293 QBI cells were transiently transfected with Flag-p62 and HA-CNOT2 plasmids and incubated for 48 h in the presence or absence of MG132 (means ± SD of 3 independent experiments, *** p < 0.001 vs untreated control by Student t test). (E) HEK293 QBI cells were transiently transfected with Flag-p62 and HA-CNOT2 plasmids and incubated for 48 h in the presence or absence of 3-MA. Cell lysates were immunoblotted with indicated antibodies (means ± SD of 3 independent experiments, ** p < 0.01; *** p < 0.001 vs untreated control by Student t test). (F) HEK293 QBI cells were transiently transfected with HA-CNOT2 plasmid or empty vector in the presence or absence of CHX. (G) HEK293 QBI cells were transiently co-transfected with HA-CNOT2, Flag-p62, and Myc-ubiquitin plasmids in the presence or absence of MG132 and 3-MA. Ubiquitination of p62 was evaluated with anti-p62/SQSTM1 antibody.

Fig 5: CNOT2 correlates with p62/SQSTM1 in HEK293 QBI cells by co-immunoprecipitation and immunofluorescence(A) HEK293 QBI cells were transiently transfected with Flag-p62 (4 μM) and/or HA-CNOT2 (4 μM). The transfected cells were treated with MG132 (10 μM) for 4 h before harvest and cell lysates were immunoprecipitated with HA-CNOT2 or Flag-p62 antibody and subjected to Western blotting. (B) HEK293 QBI cells were transiently transfected with HA-CNOT2 (4 μM) or Flag-p62 and exposed to MG132 (10 μM) for 4 h before harvest. Then cell lysates were immunoprecipiated with HA or Flag antibody and immunoblotted with antibodies of HA, Flag, p62, CNOT2 and actin. (C) HEK293 QBI cells were transiently transfected with HA-CNOT2 and/or Flag-p62 WT and Flag-p62 mutant S351 plasmids and treated with 10 μM MG132 for 4 h before cell harvest. Then cell lysates were immunoprecipitated with Flag-p62 antibody and subjected to Western blotting with antibodies of Flag and HA or Actin. (D) Immunoprecipitation of endogeneous CNOT2 with p62/SQSTM1 antibody. H1299 cells were treated 10 μM of MG132 for 4 h, cell lysates were precleaned with protein G/A beads and subsequently incubated for 1-2 h with protein G/A beads covalently coupled with anti-IgG and anti-p62/SQSTM1 antibody. (E) CNOT2 was colocalized with p62/SQSTM1. H1299 cells were transiently transfected with Flag-p62 (4 μg) and HA-CNOT2 (4 μg) plasmids (lower panel), followed by immunocytochemistry. The cells were stained with anti-p62 (green) and anti-CNOT2 (red). Bars, 10 μm.