Fig 1: p46Shc inhibition decreases CRT cell membrane translocation and alcohol-mediated induction of eukaryotic translation initiation factor 2 subunitα(EIF2S1), caspase 8 cleavage, and activation of Bak and Bax. (A) Mouse primary hepatocytes were transfected with the p46ShcẟSH2 plasmid or mock-transfected for 24 hours, exposed to alcohol (100 mmol/L for 8 hours), and immunofluorescence was performed to visualize CRT. In mock-transfected, alcohol-exposed cells, CRT showed an increased cell membrane signal (white arrows), but not in (B) p46ShcẟSH2-transfected cells (red, calreticulin; blue, 4′,6-diamidino-2-phenylindole [DAPI]). Scale bar: 20 μm. (C) In p46ShcẟSH2-transfected cells subjected to Western blot analysis, phos-EIF2S1 (Ser51), cleaved caspase 8, BAP31, Bak, and Bax were attenuated. VL-17A cells were transfected with control siRNA (siCont) or BAX siRNA (siBAX) for 24 hours, exposed to alcohol (100 mmol/L for 24 hours), and immunofluorescence was performed to visualize calreticulin. (D) In alcohol-exposed cells, CRT showed an increased cell membrane signal (white arrows) in siCont- but not in siBAX-transfected cells (red, calreticulin; blue, DAPI). Scale bar: 20 μm. (E) The percentage of surface CRT+ cells was quantified (data are presented as means ± SEM, n = 4–7 10× fields per group, ∗∗P < .05).
Fig 2: Anti-apoptotic BCL-xL and pro-apoptotic BAK proteins exhibit opposite trends during pancreatic specification from human pluripotent stem cells.a Schematic showing 17D differentiation protocol used to generate early pancreatic progenitors. Respective growth factors used are depicted at each time point. b Trypan blue staining showing the % of cell death over the course of 17D differentiation. Error bars indicate standard deviation of three biological replicates undergoing independent differentiations. Asterisk (*) indicates P < 0.05 compared between D5 and D7. Western blot showing the expression of BCL-2 proteins over the course of 17D differentiation in c H9 hESCs and d iAGb hiPSCs. e Immunofluorescence staining for BCL-xL protein on D3 and D12 cells. Scale bar represents 50 µm. f Western blot showing the expression of BAK protein upon overexpression of BCL-xL in undifferentiated hPSCs. “See also Fig. S1”.
Fig 3: DAPk1 facilitates Bik-induced cell death by forming a complex with Bik, ERK1/2 and Bak. a Protein lysates from HAECs 24 h after infection with HA-Ad-Bik or HA-Ad-BikL61G were immunoprecipitated with anti-pERK1/2 antibodies. The flow through (FT) and the immunoprecipitates (IPs) were probed with antibodies to HA, DAPK1, p-ERK1/2, and ERK1/2 by western blotting. b 293T cells were transfected with Flag-DAPk1 and 48 h later infected with 100 MOI HA-Ad-Bik or HA-Ad-BikL61G. After 24 h, protein lysates were immunoprecipitated with α-Flag antibodies and the FT and IP were probed for activated Bak, HA, and ERK1/2 by western blotting. c 293T cells transfected with various GFP-tagged DAPk1 deletions constructs, infected with HA-Ad-Bik, and subjected to immunoprecipitation using anti-HA or anti-GFP antibodies. The flow through (FT) and the IPs were probed for GFP, HA, activated Bak. d Mutation of Lys42 in the kinase domain of DAPk1 diminishes Bik–Bak–DAPk1–ERK1/2 interaction. 293T cells were transfected with Flag-DAPk1 or Flag-DAPK1K42A and 48 h later infected with 100 MOI HA-Ad-Bik. Protein lysates were immunoprecipitated using α-Flag antibodies and the FT and IP were probed for Flag, HA, Bak, and ERK1/2 by western blotting. e Cell viability of 293T cells transfected with Flag-DAPk1 or Flag-DAPK1K42A was analyzed by trypan blue assay. f, g Knockdown of DAPk1 suppresses the interaction between Bak, Bik, and ERK1/2. Protein lysates from HAECs stably expressing shCtr or shDAPk1 were infected with HA-Ad-Bik and immunoprecipitated using anti-pERK1/2 f or anti-active Bak (Ab1) g antibodies. The FT and IPs were probed for activated Bak, HA, DAPk1, and p-ERK1/2 by western blotting. h HAECs stably expressing shCtr or shDAPk1 were infected with 100 MOI of Ad-Bik in the presence of 20 µM pan-caspase inhibitor Q-VD-Oph for 18 h, fixed and stained for cytochrome c, and the percentage of cells with cytochrome c release was analyzed by fluorescent microscopy. i, j Cell lysates from HAECs stably expressing shCtr or shDAPk1 and treated with 50 ng/ml IFN-γ i or 100 MOI Ad-Bik j were analyzed for cleaved caspase 3 by immunoblotting. k HAECs stably expressing shCtr or shDAPk1 were analyzed for knockdown of DAPk1 by western blotting. Cell viability was determined by trypan blue exclusion assay 24 h after infection with100 MOI Ad-Bik. l 293T cells were transfected with plasmids expressing empty vector (EV) or ER-targeted Bcl-2 (Bcl-2b5). Forty-eight hours later, cells were infected with 100 MOI Ad-Bik and protein lysates were analyzed for the expression of Bcl-2, Bak, and Bik. Cell viability was determined by trypan blue exclusion. m 293T cells were transfected with plasmids expressing empty vector or Bcl-2b5 together with plasmids expressing Flag-DAPk1 and 48 h later infected with 100 MOI HA-Ad-Bik. Protein lysates were immunoprecipitated using α-Flag antibodies and the FT and IP were probed for Flag-DAPk1, HA-Bik, Bak and ERK1/2 by western blotting. Differences between two groups were assessed for significance by Student’s t test. ANOVA was used to perform pair-wise comparison of the data from more than two groups followed by Fisher least significant difference test. Error bars indicate ± SEM, n = 5; * = P < 0.05, ** = P < 0.01
Fig 4: A schematic depicting that Bik fulfills several functions to facilitate cell death. (1) Bik dissociates Bak/Bcl-2 and interacts with DAPk1 to form a complex with Bak that forms multimers and anchors DAPk1 to the ER to increase the contact sites of ER and mitochondria, (2) Bik disrupts the interaction between Bcl-2 and IP3R to cause calcium release, and (3) ER-associated Bak interacts with DAPk1 to facilitate mitochondrial calcium uptake
Fig 5: Schematic depiction of the proposed role of Shc and CRT translocation in alcohol-induced injury in hepatocytes. Shc induction during alcoholic hepatitis is linked to maladaptive responses with increased lipid peroxidation and production of ROS. Key to this is the mitochondrial p46Shc isoform that can inhibit fatty acid ß-oxidation. Activation of eukaryotic translation initiation factor 2 subunit α, pre-apoptotic signals with caspase-8 and BAP31 cleavage, and Bak/Bax activation lead to CRT/ERp57 complex translocation from the ER to the cell membrane where they act as DAMP and elicit an inflammatory response. Shc inhibition may protect hepatocytes against alcohol-induced injury. Created with BioRender.com.
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