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: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 4. Kisspeptin rescues DHT-induced ER stress in GT1–7 cells. (a-b) Western blots for ATF6, IRE1α, Perk, BIP p-eIF2α and eIF2α (a) and qPCR analysis of GADD34 and Xbp1s/Xbp1 (b) in GT1–7 cells 30 min after treatment with DHT (10 nM), Kp10 (a kiss1 peptide, 10 nM) or DMSO as control (ctrl). n = 3. ** P < 0.01, ***P < 0.001, t-test. (c-d) Western blots (c) or qPCR analysis (d) for ATF4, CHOP, PDI and XBP1 in GT1–7 cells 24 h after treatment with DHT (10 nM), Kp10 (10 nM), or DMSO as Ctrl. n = 3. ** P < 0.01, *** P < 0.001, t-test. (e) qPCR analysis of ATF4, BIP, PDI and CHOP in GT1–7 cells after treatment with thapsigargin (TG, an ER stress inducer, 20 nM), Kp10 (10 nM) or vehicle control (Ctrl). n = 3. *** P < 0.001, t-test. GAPDH was used as a loading control for all western blots. Error bars are smaller than symbol size except where shown.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Western Blot, Control

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

doi: 10.1016/j.bbadis.2021.166242

Figure Lengend Snippet: Fig. 5. Effect of kisspeptin on UPR in the hypo thalamus of DHT-induced PCOS rats. (a-b) qPCR analysis of PDI, ATF6, Traf2, Xbp1s/Xbp1 (a) and immunofluorescence labeling for Xbp1 (b) in the hypothalamus of DHT-induced PCOS (PCOS_DHT) rats 24 h after kp10 (100 nM,10 μl per rat) was intracerebroventricularly (i.c.v) injected at day 21 post DHT treatment. n = 3. * P < 0.05, ** P < 0.01, t-test. 3v, third ventricle. Scale bar, 20 μm. (c) qPCR analysis of BIP, PDI, Xbp1s/Xbp1 in the hypothalamus of PCOS_DHT rats 24 h after salubrinal (100 nM, 10 μl per rat) was injected (i.c.v) at day 21 post DHT treat ment. n = 4–9. * P < 0.05, t-test.

Article Snippet: Antibodies used are: IRE1a (1:500, catalog 3294, Cell Signaling Technology [CST], USA), Perk (1:500, catalog 5683, CST), BIP (1:500, catalog 3177, CST), PDI (1:500, catalog 3501,CST), Phospho-eIF2α (1:500, catalog 9721, CST), eIF2α (1:500, catalog 9722, CST), Bax (1:500, catalog 2772, CST), ATF6 (1:500, catalog 15,794–1-AP, ProteinTech, USA), ATF4 (1:500, catalog 10,835–1-AP, ProteinTech), KISS1 (1:500, catalog H-048-46PA5-106920, Thermo Fisher, USA), β-actin (1:5000, catalog A8481, Sigma-Aldrich), Xbp1 (1:500, catalog ab37152, Abcam, USA), Phospho-IRS (1:500, catalog 3203, CST), PI3K (1:500 catalog 4249, CST), Phospho-GSK-3β (1:500,catalog 5558,CST)GSK-3β (1:500, catalog 9315, CST) and GAPDH (1:5000, catalog sc-47,724, Santa Cruz, USA).

Techniques: Immunofluorescence, Labeling, Injection

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