Journal: Shock

Article Title: Hepatic Apoptosis Postburn Is Mediated by C-Jun N-Terminal Kinase 2

doi: 10.1097/shk.0b013e31827f40ab

Figure Lengend Snippet: FIG. 2. Hepatic ER stress was initiated after burn in both wild-type and JNK2 knockout (JNK2j/j) mice. Bip (A) and activated ATF6 (B) levels were quantified 1, 3, and 5 days after burn relative to "-actin. Representative blots from sham and burn groups at day 1 are shown above each graph. Downstream effectors of ER stress were transcribed in wild-type mice but not JNK2 knockout mice, as indicated by mRNA expression of XBP1s, Pdia3, and Dnajb9 (CYE) measured by real time PCR of day 5 liver samples. Data presented are mean T SEM. *P G 0.05 and ***P G 0.001 vs. sham.

Article Snippet: The antibody against mouse ATF6 was purchased from Imgenex (San Diego, Calif).

Techniques: Knock-Out, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Single-cell mRNA-regulation analysis reveals cell type-specific mechanisms of type 2 diabetes

doi: 10.1101/2023.03.23.533985

Figure Lengend Snippet: TMEM176B protein is expressed in human beta cells (A) . Reduced beta cell mass ( B ) and islet size ( C ) in Tmem176a/b double knockout (DKO) mice. Reduced acute insulin response (AIR) ( D ) and higher glucose levels calculated as iAUC ( E ) during IpGTT in Tmem176a/b DKO mice fed a high-fat diet. (F and G) Tmem176a/b DKO mice show approximately 2-fold increased actin staining in beta cells. CEBPG protein is expressed in human beta cells (H) . Reduced beta cell mass ( I ) and islet size ( J ) in Cebpg KO mice. (H) CEBPG silencing in human islets reduces the expression of ATF6, ATF4, XBP1 and DDIT3 (H) , all key mediators of UPR. Increased expression of Cebpg and decreased expression of Ins1 and Ins2 in thapsigargin-treated INS-1 832/13 cells (L) . Conversely, Ins1 and Ins2 expression was increased by thapsigargin treatment after Cebpg- silencing. The increased expression of Atf6 and Atf4 upon thapsigargin treatment was abolished after Cebpg silencing (M) . NTC, non-treated cells. Cebgp KO mice have reduced nuclear expression of ATF6 (N and O) . n=6 per group. Scale bar=50 µm. See also Fig. S4.

Article Snippet: Primary antibodies: anti-rabbit ATF6 (code NBP1-76675, dilution 1:200, Novus Biologicals, Centennial, CO), anti-rabbit CEBPG (code HPA012024, dilution 1:20, Sigma Aldrich), anti-rabbit DSTN (code ab186754, dilution 1:100, Abcam), anti-rabbit GABARAP (code HPA-78365, 1:500 dilution, Sigma Aldrich), anti-guinea pig insulin (code A0564, 1:1000 dilution, Dako, Glostrup, Denmark), anti-rabbit KIAA1324 (code PA5-67123, dilution 1:800, Life Technologies), anti-rabbit SPIRE1 (ab130403, 1:1000 dilution, Abcam), anti-rabbit TMEM176A (code NBP1-83283, 1:20 dilution, Novus Biologicals) and anti-rabbit TMEM176B (code CSB-PA023758LA01HU, 1:50 dilution, CusaBio, Houston, TX).

Techniques: Double Knockout, Staining, Expressing

Journal: Journal of Endocrinology

Article Title: Exendin-4 inhibits glucolipotoxic ER stress in pancreatic β cells via regulation of SREBP1c and C/EBPβ transcription factors

doi: 10.1530/joe-12-0311

Figure Lengend Snippet: Figure 2 Exendin-4 inhibits HG/PA-induced expression of ER stress markers. (A) INS-1 cells were treated with vehicle (CON) or HG/PA in the absence or presence of 5 nM exendin-4 (Ex) for 6 h. Total RNA was isolated and mRNA expression of sXBP1, CHOP, and Bip were determined by qRT-PCR. Cyclophilin was used as control gene. (B) INS-1 cells were treated as described earlier for 12 or 18 h and total cell extracts were analyzed for PERK, p-PERK, p-eIF2a, p-IRE1a, p-JNK, ATF6 (p50), and actin by immunoblotting using specific antibodies. (C) Quantitative analysis of western blots. Relative abundance of each band was estimated by densitometric analysis. Each bar represents the meanGS.E.M. from three independent experiments. *P!0.05.

Article Snippet: The sources of various reagents and materials were as follows: exendin-4, D-glucose, palmitate (PA), fatty acid-free BSA were from Sigma–Aldrich; anti-C/EBPb and anti-lamin B antibodies were from Santa Cruz Biotechnology, Inc.; polyclonal antibody against SREBP1 (SREBF1) was from Abcam (Cambridge, MA, USA); MAB against ATF6 was from IMGENEX(SanDiego,CA,USA); andanti-C/EBPhomologous protein (CHOP), anti-caspase-3, anti-poly(ADP-ribose) polymerase (PARP), anti-PERK, anti-phospho-PERK, anti-phosphoIRE1a, anti-phospho-c-Jun N-terminal kinase (JNK), and antiphospho-eukaryotic initiation factor 2a (eIF2a) antibodies were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Control, Western Blot

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