Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Western blot analysis results for ERS-associated proteins. (A) The representative blotting images and (B) quantified data demonstrate elevated p-PERK, eIF2a, CHOP, Bax/Bcl-2, Caspase-12 and cleaved Caspase-3 level in L539fs/47-hERG cells, which can be reversed by 4-PBA. Each experiment was repeated at least three times. # P<0.05 between the three model cell groups; * P<0.05 between two subgroups treated with or without 4-PBA intervention. P<0.05 was considered to indicate a statistically significant difference. ERS, endoplasmic reticulum stress; hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Proposed mechanisms of mutated hERG-induced cell apoptosis. Mutated L539fs/47-hERG protein retained in the ER lumen provokes ERS. Elevated expression levels of GRP78 activate the PERK-eIF2α-CHOP pathway, which is important in the mechanism of L539fs/47-hERG-induced ERS-mediated cell apoptosis. ER-specific caspase-12 is involved in the apoptotic mechanisms caused by the hERG mutation through cleaving and activating caspase-3. The pair of apoptosis factors Bax/Bcl-2 also regulate L539fs/47-hERG-induced cell apoptosis. hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; ER, endoplasmic reticulum; ERS, ER stress; ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis

Journal: Journal of advanced research

Article Title: Short-chain fatty acid butyrate against TMAO activating endoplasmic-reticulum stress and PERK/IRE1-axis with reducing atrial arrhythmia.

doi: 10.1016/j.jare.2024.08.009

Figure Lengend Snippet: Fig. 4. SB supplement ameliorated TMAO-mediated activation of ER stress signaling and dysregulation of ionic signaling. (A) and (B) Representative images and statistical data show the expression of pPERK, PERK, pIRE1a, IRE1a, pNF-jB, NF-jB, pIP3R, IP3R, NCX, Kv1.5, and b-actin in HL-1 cells from control group (n = 3), TMAO treated (n = 3), SB treated (n = 3), and TMAO combined with SB treated group (n = 3) in three independent experiments. b-Actin was used as a loading control. *P < 0.05. **P < 0.01. TMAO versus control or SB or TMAO combined with SB treatment.

Article Snippet: Then, the blots were incubated with primary antibodies against phospho-PERK (pPERK; #3179, Cell signaling), PERK (#3192, Cell signaling), phospho-IRE1a (pIRE1a; NB100-2323, Novus), IRE1a (#3294, Cell signaling Technology), phospho-NFjB (pNF-jB; #3033, Cell Signaling Technology), NF-jB (#8242, Cell Signaling Technology), phospho-IP3R (pIP3R; #3760, Cell signaling Technology), IP3R (#8568, Cell signaling Technology), NCX (MA3926, Thermo Fisher Scientific), Kv1.5 (APC-004, Alomone Lab), and b-actin (sc-47778, Santa Cruz).

Techniques: Activation Assay, Expressing, Control

Journal: Journal of advanced research

Article Title: Short-chain fatty acid butyrate against TMAO activating endoplasmic-reticulum stress and PERK/IRE1-axis with reducing atrial arrhythmia.

doi: 10.1016/j.jare.2024.08.009

Figure Lengend Snippet: Fig. 8. SB supplement ameliorated atrial fibrosis and PERK/IRE1a/NF-jB axis of TMAO-treated mice. The expression of atrial fibrosis (blue area) was detected by Masson’s staining and immunostaining of pPERK, pIRE1a, and pNF-jB in the atrium tissues derived from mice gavage with control (n = 5), TMAO (n = 5), and TMAO combined with SB (n = 5). The expressions of pPERK are indicated by hollow arrowheads. (bar, 25 lm). ** p < 0.01. TMAO treatment versus control group or TMAO combined with SB treated group. (For interpretation of the references to colour in this Fig. legend, the reader is referred to the web version of this article.)

Article Snippet: Then, the blots were incubated with primary antibodies against phospho-PERK (pPERK; #3179, Cell signaling), PERK (#3192, Cell signaling), phospho-IRE1a (pIRE1a; NB100-2323, Novus), IRE1a (#3294, Cell signaling Technology), phospho-NFjB (pNF-jB; #3033, Cell Signaling Technology), NF-jB (#8242, Cell Signaling Technology), phospho-IP3R (pIP3R; #3760, Cell signaling Technology), IP3R (#8568, Cell signaling Technology), NCX (MA3926, Thermo Fisher Scientific), Kv1.5 (APC-004, Alomone Lab), and b-actin (sc-47778, Santa Cruz).

Techniques: Expressing, Staining, Immunostaining, Derivative Assay, Control

Journal: International journal of molecular medicine

Article Title: FGF21 attenuates hypoxia‑induced dysfunction and apoptosis in HPAECs through alleviating endoplasmic reticulum stress.

doi: 10.3892/ijmm.2018.3705

Figure Lengend Snippet: Figure 4. FGF21 alleviates the hypoxia‑induced ERS by modulating the expression of ERS‑related proteins in HPAECs. HPAECs were treated as indicated and protein expression levels were examined by western blotting. Representative blots and quantification are shown for (A) BiP, (B) p‑PERK and PERK, (C) the ratio of p‑PERK/PERK, (D) CHOP, (E) Bcl‑2. (F) Relative expression of the ERS‑dependent apoptotic protein caspase‑4 in HPAECs was analysed by western blotting. GAPDH was used as an internal control. All experiments were performed in triplicate, and data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. N group; ##P<0.01 vs. the H group; &&P<0.01 vs. the H+T group. FGF21, fibroblast growth factor 21; ERS, endoplasmic reticulum stress; HPAECs, human pulmonary arterial endothelial cells; BiP, binding immunoglobulin protein; p‑, phosphorylated; PERK, protein kinase R‑like endoplasmic reticulum kinase; CHOP, transcrip- tion factor C/EBP homologous protein; Bcl‑2, B cell lymphoma-2; N, normoxia; H, hypoxia; F, FGF21; S, salubrinal; T, tunicamycin.

Article Snippet: Rabbit antibodies against phosphorylated (p-) PERK were purchased from Biorbyt (cambridge, UK).

Techniques: Expressing, Western Blot, Control, Standard Deviation, Binding Assay

Journal: International journal of molecular medicine

Article Title: FGF21 attenuates hypoxia‑induced dysfunction and apoptosis in HPAECs through alleviating endoplasmic reticulum stress.

doi: 10.3892/ijmm.2018.3705

Figure Lengend Snippet: Figure 6. Effect of FGF21 on NO and ET‑1 secretion in HPAECs. (A) HPAECs were treated as indicated and at the end of treatment, the cell culture medium was collected and assayed by ELISA for the levels of secreted NO and (B) the levels of secreted ET‑1. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. the N group; #P<0.05 and ##P<0.01 vs. the H group; &&P<0.01 vs. the H+T group. (C) Schematic of the proposed mechanism by which FGF21 attenuates hypoxia‑induced apoptosis and dysfunction by alleviating ERS in HPAECs. FGF21, fibroblast growth factor 21; NO, nitric oxide; ET‑1, endothelin‑1; HPAECs, human pulmonary arterial endothelial cells; ERS, endoplasmic reticulum stress; N, normoxia; H, hypoxia; F, FGF21; S, salubrinal; T, tunicamycin; BiP, binding immunoglobulin protein; p‑, phosphorylated; PERK, protein kinase R‑like endoplasmic reticulum kinase; CHOP, transcription factor C/EBP homologous protein; Bcl‑2, B cell lymphoma-2.

Article Snippet: Rabbit antibodies against phosphorylated (p-) PERK were purchased from Biorbyt (cambridge, UK).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Binding Assay

Journal: Biomedicines

Article Title: Dyskerin Downregulation Can Induce ER Stress and Promote Autophagy via AKT-mTOR Signaling Deregulation

doi: 10.3390/biomedicines10051092

Figure Lengend Snippet: Dyskerin depletion induces ERS and increases UPR markers. ( A ) Confocal images showing the dramatic increase in GRP78/BiP (red or Fire-Lut) expression in RKO-shDKC1 cells treated with Dox for 96 h (Dox+, right panel) compared to untreated control cells (Dox–, left panel). Dyskerin (green or Thal-Lut) downregulation is evident from the strong reduction in the relative signal in Dox+ cells. Nuclei were counterstained with DAPI. GRP78/BiP and dyskerin levels were quantified in multiple cells ( n = 18 RKO-shDKC1 Dox–; n = 20 RKO-shDKC1 Dox+ 96 h), normalized and reported relative to the Dox– condition as mean ± SEM. Scale bars 10 μm. ( B , C ) Western blot analysis confirming the upregulation of GRP78/BiP and showing the induction of key components of the PERK branch of UPR in RKO-shDKC1 ( B ) and HEK 293T-shDKC1 ( C ) Dox–treated (Dox+) cells for 48 or 96 h. Protein levels were normalized to GAPDH. Data are shown as mean ± SEM ( n = 3). All statistical analyses were performed using an unpaired one-tailed t -test in pairwise comparisons with respect to the Dox– condition (**** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, p > 0.05).

Article Snippet: The following primary antibodies were used in this study: dyskerin (Elabscience Biotechnology Inc., Houston, TX, USA, E-AB-31251; 1:100), GAPDH (Abcam, CA, USA, ab56788; 1:100), GRP78/BiP (Thermo Fisher Scientific, Waltham, MA, USA, PA5-11418; 1:1000), phospho PERK (Elabscience Biotechnology Inc., Houston, TX, USA, E-AB-21296; 1:1000), phospho eIF2α (Cell Signaling Technology (CST), Heidelberg, Germany, 9721; 1:1000), ATF4 (Santa Cruz Biotechnology; 1:500), CHOP (DSHB—Developmental Studies Hybridoma Bank, University of Iowa, Iowa city, USA; CPTC-DDIT3-1-S; 1:500), HSP70 (Santa Cruz biotechnology, sc-25837; 1:1000), HSP90 (Stressgen Biotechnologies Corporation, Ann Arbor, MI, USA, SPA-846; 1:1000), LC3 (Abcam, CA, USA, ab51520; 1:2000), Beclin 1 (Elabscience Biotechnology Inc., Houston, TX, USA, E-AB-33743; 1:1000), PARP (Santa Cruz Biotechnology, Dallas, TX, USA, sc-7150; 1:1000), BAX (Cell Signaling Technology (CST), Heidelberg, Germany2772; 1:500), phospho 4E-BP1 (Cell Signaling Technology (CST), Heidelberg, Germany, 2855; 1:1000), phospho p70 S6K (Cell Signaling Technology (CST), Heidelberg, Germany9205; 1:1000), phospho AKT (Cell Signaling Technology (CST), Heidelberg, Germany, 9271; 1:1000), and phospho GSK3β (GeneTex, Inc. (North America), Alton Pkwy Irvine, CA, GTX-59576; 1:1000).

Techniques: Expressing, Western Blot, One-tailed Test