Fig 1: The Livin-H2A.XY142ph axis activated autophagy in colon cancer cells through transcriptionally regulating ATGs. (A) Livin−/− SW480 cells were transfected with H2A.XY142A or H2A.XY142F mutant which were similar with the phosphorylated formation of H2A.XY142. After starvation, the mRNA levels of ATG5 and ATG7 were measured by RT-qPCR, and the protein levels of ATG5, ATG7, H2A.X and Livin were tested by western blotting. Data were shown as (Mean ± SD). **P < 0.01, ***P < 0.001; (B) The pGL3-ATG5 and pGL3-ATG7 plasmid were constructed for the luciferase assay. The WT SW480 cells were co-transfected with pGL3-ATG5/ pGL3-ATG7 plasmid and H2A.XWT/H2A.XY142F plasmid, then the average relative lights units were used to evaluate gene transcriptional activity. Data were shown as (Mean ± SD). **P < 0.01, ***P < 0.001. The protein levels of ATG5, ATG7, H2A.X, Livin and LC3I/LC3II were tested by western blotting.
Fig 2: Livin directly interacts with H2A.X and phosphorylates it at Y142. (A) The interaction of Livin with H2A.X was demonstrated by Immunoprecipitation in SW480 cells, the binding of H2A.X and Livin was detected by western blotting; (B) HCT116 cells transduced with shRNA targeting 3′-UTR of H2A.X and selected using puromycin. Selected cells were immunoblotted to confirm knockdown of H2A.X. (C) HCT116 cells harboring shRNA-H2A.X were transfected with H2A.XWT or H2A.XY142F plasmid tagged with HA. The immunoprecipitation was performed and the bonding band of H2A.XWT/ H2A.XY142F and Livin was tested by western blotting; (D) Coomassie stain to confirm effective expression and purification of GST-Livin and His-H2A.X; (E) in vitro pull-down assay was done to test the association between the Livin plasmid tagged with GST and His-H2A.X; (F) in vitro kinase assay using recombinant Livin and wild-type or Y142F mutant H2A.X. HA, human influenza hemagglutinin; GST, Glutathione S-transferase; WT, wild type; H2A.XY142ph, H2A.XY142 phosphorylation.
Fig 3: H2A.XY142ph was necessary for Livin-mediated autophagy in starvation-stimulated colon cancer cells. (A,B) H2A.X−/−Livin+/+ SW480 cells were co-transfected with H2A.XWT/H2A.XY142F plasmid and LC3 EGFP-tagged plasmid. After starvation, the fluorescence of EGFP-LC3 puncta was measured, 80X, scale bar = 50 μm. Each field was randomly selected for the statistics of fluorescence intensity, and the average of 6 fields of view was calculated. Data were shown as (Mean ± SD). ***P < 0.001. The protein levels of total H2A.X and Livin was measured by western blotting (B); (C) The kinase activity of Livin for H2A.XY142 in SW480 cells with starved stimulation for 30 min and 1 h was evaluated by protein levels of H2A.XY142ph; (D,E) WT SW480 cells were transfected with Livin plasmid to overexpress Livin (Livin+) after starvation. The fluorescence of EGFP-LC3 puncta was measured, 80X, scale bar = 50 μm. Each field was randomly selected for the statistics of fluorescence intensity, and the average of 6 fields of view was calculated. Data were shown as (Mean ± SD). ***P < 0.001. The protein levels of H2A.XY142ph, H2A.X and Livin were measured by western blotting (E).
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