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: Frontiers in Pharmacology

Article Title: Coreopsis tinctoria Nutt. attenuates ultraviolet A photodamage by suppressing endoplasmic reticulum stress-induced apoptosis via Nrf2 crosstalk

doi: 10.3389/fphar.2025.1686234

Figure Lengend Snippet: Extraction from CT decreases UVA-induced ER stress in HaCaT cells. HaCaT cells were subjected to UVA (5 J/cm 2 ) radiation and then treated with the indicated concentrations of extraction from CT. (A) Western blot analysis was performed to determine the protein levels of phosphorylated IRE1 (pIRE1), ATF6, phosphorylated PERK (p-PERK), phosphorylated eIF2a (p-eIF2a) and CHOP in different treatments. (B) Immunofluorescence assay was performed to detect CHOP expression. (C) Representative images showing the ER morphology in control, UVA-treated and CT-treated cells with UVA exposure. Cells were stained with ER tracker in green and counterstained with DAPI for nuclear visualization. N = 4. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: IRE1 (phospho S724) (Abcam, ab124945); CHOP (Proteintech,15204-1-AP); NRF2 (Proteintech, 16396-1-AP); ATF6 (Proteintech, 24169-1-AP); PERK (phospho Thr982) (Proteintech, 82534-1-RR); eIF2α(phospho Ser51) (Proteintech, 68023-1-Ig); GADD34 (Proteintech, 10449-1-AP); Caspase9 (Proteintech, 10380-1-AP); Cleaved caspase3 (Proteintech, 25128-1-AP); JNK (Proteintech, 51153-1-AP); JNK (phospho Tyr185) (Proteintech, 80024-1-RR); Bax (Proteintech, 50599-2-Ig); BCL2 (Proteintech, 60178-1-Ig); HO-1 (Proteintech, 10701-1-AP); β-Actin (Proteintech, 20536-1-AP); Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse secondary antibodies were applied (Jackson, 111-035-003 and 115-035-003).

Techniques: Extraction, Western Blot, Immunofluorescence, Expressing, Control, Staining

Journal: Frontiers in Pharmacology

Article Title: Coreopsis tinctoria Nutt. attenuates ultraviolet A photodamage by suppressing endoplasmic reticulum stress-induced apoptosis via Nrf2 crosstalk

doi: 10.3389/fphar.2025.1686234

Figure Lengend Snippet: CT alleviates UVA-induced ER stress and apoptosis via Nrf2 in HaCaT cells. (A) HaCaT cells were subjected to UVA radiation and treated with 50 or 100 μg/mL CT and/or siNrf2. Western blot assays were performed to detect the levels of ATF6, phosphorylated elF2a (p-elF2a), CHOP, GADD34, Cleaved caspase9, Cleaved caspase3, JNK, phosphorylated JNK (p-JNK), Bax, BCL2, cytoplasmic and nuclear NRF2 and HO-1. (B) Flow cytometry analysis was performed to determine cell apoptosis. Quantification of the percentage of apoptotic cells in different treatments. (C) Heatmap generated the data from RNAseq. Genes are all related to NRF2 downstream anti-oxidant or detoxification target genes. The figure was generated by SRplot . N = 4. * p ≤ 0.05; ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The membranes were blocked with 5% non-fat milk and incubated with the following primary antibodies: IRE1 (phospho S724) (Abcam, ab124945); CHOP (Proteintech,15204-1-AP); NRF2 (Proteintech, 16396-1-AP); ATF6 (Proteintech, 24169-1-AP); PERK (phospho Thr982) (Proteintech, 82534-1-RR); eIF2α(phospho Ser51) (Proteintech, 68023-1-Ig); GADD34 (Proteintech, 10449-1-AP); Caspase9 (Proteintech, 10380-1-AP); Cleaved caspase3 (Proteintech, 25128-1-AP); JNK (Proteintech, 51153-1-AP); JNK (phospho Tyr185) (Proteintech, 80024-1-RR); Bax (Proteintech, 50599-2-Ig); BCL2 (Proteintech, 60178-1-Ig); HO-1 (Proteintech, 10701-1-AP); β-Actin (Proteintech, 20536-1-AP); Horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse secondary antibodies were applied (Jackson, 111-035-003 and 115-035-003).

Techniques: Western Blot, Flow Cytometry, Generated

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mfn2 Regulates High Glucose-Induced MAMs Dysfunction and Apoptosis in Podocytes via PERK Pathway

doi: 10.3389/fcell.2021.769213

Figure Lengend Snippet: ER stress-related protein expression and apoptosis in glomeruli from diabetic rats and HG-treated cultured podocytes. (A–G) Representative Western blots of glomerular CHOP, cleaved caspase3, p-PERK, PERK, IRE1α, ATF6β expression and quantification per group (n = 3). * p < 0.05 relative to control. (H–L) Representative Western blots of CHOP, cleaved caspase3, p-PERK, PERK, IRE1α, ATF6β expression in podocytes cultured with different medium and quantitation per group (n = 3). * p < 0.05 compared with podocytes cultured in normal conditions. (M,N) Flow cytometry analysis of apoptosis in cultured podocytes and quantitation per group (n = 3). * p < 0.05 compared with podocytes cultured in normal conditions. CTL, control; STZ, streptozotocin; N = 5 mM glucose for 24 h; MA = 5 mM glucose+20 mM mannitol for 24 h; HG1 and HG2 = 25 mM glucose for 24 and 36 h; NS, not significant.

Article Snippet: The following primary antibodies were used in this study: Mfn2 (ab123773; 1:1,000; Abcam, United Kingdom); cleaved caspase3 (ab32042; 1:1,000; Abcam, United Kingdom); CHOP (sc-7351; 1:500; Santa Cruz, United States); Bcl2 (ab32124; 1:1,000; Abcam, United Kingdom); Bax (60267-1-lg; 1:1,000; Proteintech, United States); IRE1α (#3294; 1:1,000; CST, United States); ATF6β (15794-1-AP; 1:1,000; Proteintech, United States); p-PERK (#3719; 1:1,000; CST, United States); PERK (sc-377400; 1:500; Santa Cruz, United States); Myc-tag (#562; 1:1,000; Medical & Biological Laboratories CO. LTD., Beijing, China); GAPDH (sc-32233; 1:2000; Santa Cruz, United States).

Techniques: Expressing, Cell Culture, Western Blot, Control, Quantitation Assay, Flow Cytometry

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