Journal: Antioxidants

Article Title: CHIP Haploinsufficiency Exacerbates Hepatic Steatosis via Enhanced TXNIP Expression and Endoplasmic Reticulum Stress Responses

doi: 10.3390/antiox12020458

Figure Lengend Snippet: CHIP is involved in unfolded protein responses (UPRs) and apoptosis in hepatocytes. ( a ) AML12 cells were transfected with control (siControl) or CHIP siRNA (siCHIP) for 48 h and then treated with tunicamycin (TM, 5 or 10 μM) for 24 h. Protein levels of GRP78, ATF6, XBP-1s and CHIP were determined by immunoblotting. A-tubulin was used as a loading control. ( b ) AML12 cells were transfected with siControl or siCHIP for 48 h and then treated with TM (10 μM) for 24 h. Protein levels of cleaved PARP-1, cleaved caspase-3 (cleaved Casp3), and CHIP were determined by immunoblotting. α-tubulin was used as a loading control. ( c ) AML12 cells were transfected with siControl or siCHIP for 48 h and then treated with TM (10 μM) for 24 h. Expression of UPR-related genes were measured by qRT-PCR. Relative expression levels were normalized to GAPDH levels. ** p < 0.01 vs. siControl, ## p < 0.01 vs. siCHIP, † p < 0.05 and †† p < 0.01. ( d ) Primary hepatocytes from CHIP +/+ , CHIP +/− , and CHIP −/− mice were treated with TM (2 or 10 μM) for 6 h. Protein levels of GRP78, CHOP, XBP-1s, and CHIP were measured by immunoblotting. β-actin was used as a loading control. ( e ) Primary hepatocytes from CHIP +/+ and CHIP +/− mice were treated with brefeldin A (BFA, 1 or 2 μM) for 6 or 9 h. Protein levels of GRP78, CHOP, cleaved PARP-1, and CHIP were measured by immunoblotting. α-tubulin was used as a loading control.

Article Snippet: The antibodies were purchased from the following vendors: TXNIP (MBL International, Woburn, MA, USA); KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany); XBP-1s (BioLegend, San Diego, CA, USA); ATF6 (Novus Biologicals, Littleton, CO, USA); ATF4, GADD153 (CHOP), HA, and CHIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Akt, p-Akt, ACC, FAS, PARP-1, and cleaved Caspase-3 (Cell Signaling, Danvers, MA, USA); NLRP3 (ThermoFisher, Waltham, MA, USA); PGC1α (abcam, Cambridge, UK); and α-tubulin and β-actin (Sigma Aldrich, St. Louis, MO, USA).

Techniques: Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR, ChIP-chip

Journal: PLoS Biology

Article Title: Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome

doi: 10.1371/journal.pbio.1002511

Figure Lengend Snippet: (A) Neurons were transfected with plasmids expressing scrambled shRNA or Wfs1 shRNA, firefly luciferase constructs containing ATF6 or ATF4 binding sites or a XBP-1 splicing reporter, and Renilla luciferase. Firefly luciferase signal normalized to Renilla signal demonstrates a moderate increase in ATF6 and ATF4 reporter activity. (B) Positive control experiments in which the above-mentioned reporter systems and Renilla luciferase were co-transfected with ATF4, ATF6, or IRE1. (C) The mitochondrial fusion rate is reduced by Wfs1 shRNA and is restored by co-expressing wt HSPA5 ( p = 0.001 for interaction, two-way ANOVA). (D) ATPase-deficient HSPA5 mutant (T37G) but not peptide binding-deficient mutant (P495L) restores the fusion rate reduced by Wfs1 shRNA. (E) HSPA5 overexpression attenuates mitophagy activated by Wfs1 silencing ( p = 0.012 for interaction). (F–H) Activation of the primary ER stress pathways by overexpression of ATF6, ATF4, or IRE1 modulates neither fusion rate (F), mitochondrial length (G), nor mitophagy (H). (I–L) Silencing of ATF6 or ATF4 modulates neither fusion rate (I, J) nor mitophagy (K, L). * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with respective control groups, or ### p < 0.001 compared with the Wfs1 shRNA-transfected control group and ns non-significant compared with the Wfs1 shRNA-transfected control group. Underlying data is shown in .

Article Snippet: ATeam (51958), ATF6 (11975), ATF6-GL3 (11976), ATF4 (26114), ATF4-luc (21850), WFS1 wt (13011), WFS1 P724L (13012), IRE1α (13009), D1ER (36325), D3cpv (36323), DRP1 K38A, EGFP-LC3 (24920), HSPA5 wt (27164), HSPA5 T37G (27165), HSPA5 P495L (27166), NRF2 (21555), ORAI1 (21638), PSD-95 (15463), and pAAV-hSyn-DsRedExpress (22907) were obtained from Addgene (Cambridge, MA).

Techniques: Transfection, Expressing, shRNA, Luciferase, Construct, Binding Assay, Activity Assay, Positive Control, Mutagenesis, Over Expression, Activation Assay, Control

Journal: Journal of Experimental & Biomedical Sciences/Biomedical Science Letters

Article Title: Expression of Beta-catenin-interacting Protein 1 (CTNNBIP1) Gene Is Increased under Hypothermia but Decreased under Additional Ischemia Conditions

doi: 10.15616/bsl.2014.20.3.168

Figure Lengend Snippet: Fig. 3. Effect of hypothermia on gene expression of ER chaperones (A), XBP1 mRNA splicing (B), ER stress sensors (C), apoptosis- associated factors (D) and autophage-associated factors (E). PC12 cells were incubated under control conditions (37 ) or hypothermia ℃ (32 ) for 1 day. Gene expressions were determined by RT ℃ -PCR by conditions described in Materials and methods (A, D, E). The product of XBP1-Pst1 digestion was subjected to electrophoresis on the agarose gel (B). Both cleavage of ATF-6 and phosphorylation elF2α were assessed by Western blotting (C). The experiments were performed thrice and results represent the average and representative findings are shown. Bip; binding immunoglobulin protein, Canx; calnexin, PDI; protein disulfide isomerase, ATF6; activating transcription factor 6, phosph-elF2α; phosphorylation of eIF2α, Bak1; BCL2-antagonist/killer 1, Bcl-2; B-cell lymphoma 2, LC3; microtubule-associated protein 1 light chain 3α, beclin; coiled-coil myosin-like BCL2-interacting protein.

Article Snippet: Mouse anti-ATF6 antibody was obtained from Imgenex (San Diego, CA, USA).

Techniques: Gene Expression, Incubation, Control, Electrophoresis, Agarose Gel Electrophoresis, Phospho-proteomics, Western Blot, Binding Assay