Journal: Cell death & disease

Article Title: Bax Inhibitor-1 preserves pancreatic β-cell proteostasis by limiting proinsulin misfolding and programmed cell death.

doi: 10.1038/s41419-024-06701-x

Figure Lengend Snippet: Fig. 5 BI-1 deletion increases cell death and leads to ER stress and activated inflammasome markers in human β-cells. A Cell death was quantified in the human β−cell line EndoC-βH1 cells transfected with control (siCrtl) or BI-1 siRNA (siBI-1) in response to chemical ER stress (Thapsigargin 1 μM; Tunicamycin 5 μg/ml) compared to normal media. n = 4. $P ≤0.05; $$P ≤0.01; $$$P ≤0.001. $$$$P ≤0.0001. $ represents differences with control. *Represents differences with indicated treated conditions, **P ≤0.01; ****P ≤0.0001. B Cell death was quantified in EndoC-βH1. The concentrations of chemicals were used as following: z-VAD-FMK (50 μmol/L, 30 min pre-incubation), Necrostatin-1 (Nec-1) (10 μmol/L, thapsigargin (1 μmol/L), or an equal volume of DMSO (Sigma-Aldrich). $ represents differences with control. *Represents differences with indicated treated conditions, **P ≤0.01; ****P ≤0.0001. n = 4–7. C Western blotting analysis of phospho-IRE1α, sXBP1, active- caspase-1, and pro-IL-1β protein levels assessed from EndoC-βH1 cells transfected with control or BI-1 siRNA prior to treatment (n = 4–7).

Article Snippet: After transfection, the cells were then treated with the indicated concentrations of z-VAD-FMK (50 μmol/L, 30 min pre-incubation, Selleck, Munich, Germany), Nec-1 (10 μmol/L, MedChemExpress, Sollentuna, Sweden), Thapsigargin (1 μM, Sigma-Aldrich), or an equal volume of DMSO (Sigma-Aldrich), respectively, as specified.

Techniques: Transfection, Control, Incubation, Western Blot

Journal: Molecular oncology

Article Title: NFAT1 Promotes Intratumoral Neutrophil Infiltration by Regulating IL8 Expression in Breast Cancer

doi: 10.1016/j.molonc.2015.02.004

Figure Lengend Snippet: NFAT1 promotes the expression of IL8. A, MDA‐MB‐231 cells transduced with active NFAT1 (doxycycline 200 ng/ml, 48 h) and IL8 mRNA measured by RT‐qPCR, normalized to 18S. B–C, MDA‐MB‐231 cells transduced with active NFAT1 (doxycycline 100 or 400 ng/ml, 72 h), and IL8 expression determined by immunoblot (B; lys: lysate; CM: conditioned media; *denotes an unspecific band) and ELISA (C; n = 3). D–E, MDA‐MB‐231 cells transduced with NFAT1 shRNA #2 or vector control, and treated with vehicle or thapsigargin for 24 h, and IL8 expression assed by RT‐qPCR (D) or immunoblotting (E; CM: conditioned media). F, indicated triple‐negative cell lines were pre‐treated with cyclosporin A (CsA, 1 μM, 1 h) or vehicle, and then treated with thapsigargin (thapsi, 50 nM) for 24 h, after which conditioned media was filtered and used for ELISA to measure IL8. G, the indicated cells lines were pre‐treated with cyclosporin A (CsA, 1 μM, 24 h) or vehicle and thapsigargin (thapsi, 200 nM, 24 h), and lysates immunoblotted with the indicated antibodies. Statistical significance was determined by Student's unpaired t‐test. *p < 0.05; **p < 0.01; ***p < 0.001. All results are representative of at least 3 independent experiments.

Article Snippet: Cells were treated with 200 nM thapsigargin (Santa Cruz Biotechnologies, Dallas, TX) for 20 h. Where indicated, cells were pretreated with 1 μM cyclosporine A (Sigma–Aldrich, St. Louis, MO) for 1 h. After treatments, cells were harvested, and conditioned media was collected and filtered through 0.45 μm filters.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, shRNA, Plasmid Preparation, Control

Journal: Molecular oncology

Article Title: NFAT1 Promotes Intratumoral Neutrophil Infiltration by Regulating IL8 Expression in Breast Cancer

doi: 10.1016/j.molonc.2015.02.004

Figure Lengend Snippet: NFAT1 regulates the transcription of the IL8 gene by associating with the proximal promoter. A, Figure depicts a proximal segment of the endogenous IL8 promoter 1.4 kb upstream of translation start site, along with the sequence of the conserved NFAT consensus binding motif and the IL8‐luc 4Xmut reporter, in which crucial NFAT‐binding bases have been mutated. B, 293T cells were transfected with the IL8‐luc wt or IL8‐luc 4Xmut reporter along with either pcDNA3‐HA‐NFAT1 CA or pcDNA3 vector. Luciferase activity was normalized to beta‐galactosidase activity. C, MDA‐MB‐231 cells treated with vehicle, thapsigargin or cyclosporin A were lysed, cross‐linked and NFAT1 immunoprecipitated, followed by PCR using primers for IL8 promoter or actin coding region and IL8 coding region to control unspecific binding. Statistical significance was determined by Student's unpaired t‐test. *p < 0.05; **p < 0.01; ***p < 0.001. All results are representative of at least 2 independent experiments.

Article Snippet: Cells were treated with 200 nM thapsigargin (Santa Cruz Biotechnologies, Dallas, TX) for 20 h. Where indicated, cells were pretreated with 1 μM cyclosporine A (Sigma–Aldrich, St. Louis, MO) for 1 h. After treatments, cells were harvested, and conditioned media was collected and filtered through 0.45 μm filters.

Techniques: Sequencing, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Immunoprecipitation, Control

Journal: Scientific Reports

Article Title: ERS regulates endometrial epithelial cell autophagy through XBP1s -mediated activation of the PI3K/AKT pathway

doi: 10.1038/s41598-024-84461-6

Figure Lengend Snippet: XBP1s knockdown reversed the effect of TG on gEECs autophagy. The experimental treatment conditions were as follows: pretreatment with TG for 2 h, followed by rapamycin treatment 24 h. (A-D) The relative protein expression of SQSTM1, ATG5, and the LC3II/LC3I were analyzed using western blotting and were quantified by densitometry. (E-F) The relative SQSTM1 and ATG5 levels were quantified through RT-qPCR. Data is represented as the means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control group. # , P < 0.05; ## , P < 0.01; ### , P < 0.001 vs. other group.

Article Snippet: The medium was replaced with fresh medium and the following treatments were initiated: (1) rapamycin (50 nM; TargetMol, Boston, MA, USA) for 3 h, 6 h, 12 h, and 24 h; (2) pretreatment with TG (Thapsigargin; 50 nM; an ERS activator; TargetMol) or 4-PBA (4-Phenylbutyric acid; 1 mM, an ERS inhibitor; Abcam, Cambridge, UK) or SC79 (10 µM; a PI3K/AKT pathway activator; TargetMol) or LY294002 (10 µM, a PI3K/AKT pathway inhibitor; TargetMol) for 2 h, followed by rapamycin treatment.

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

Journal: Scientific Reports

Article Title: ERS regulates endometrial epithelial cell autophagy through XBP1s -mediated activation of the PI3K/AKT pathway

doi: 10.1038/s41598-024-84461-6

Figure Lengend Snippet: Effect of PI3K/AKT pathway inhibition by LY294002 on overexpression of XBP1s. The experimental treatment conditions were as follows: pretreatment with LY294002 for 2 h, followed by rapamycin treatment 24 h. (A-D) Quantification of SQSTM1, ATG5, and the LC3II/LC3I band intensities from three independent experiments as determined using densitometric analysis. (E-F) SQSTM1 and ATG5 levels were detected using RT-qPCR. (G) Immunofluorescence images of LC3B expression in gEECs. Representative images of three independent experiments are shown. Scale bar = 10 μm. Data is represented as the means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control group. # , P < 0.05; ## , P < 0.01; ### , P < 0.001 vs. other group.

Article Snippet: The medium was replaced with fresh medium and the following treatments were initiated: (1) rapamycin (50 nM; TargetMol, Boston, MA, USA) for 3 h, 6 h, 12 h, and 24 h; (2) pretreatment with TG (Thapsigargin; 50 nM; an ERS activator; TargetMol) or 4-PBA (4-Phenylbutyric acid; 1 mM, an ERS inhibitor; Abcam, Cambridge, UK) or SC79 (10 µM; a PI3K/AKT pathway activator; TargetMol) or LY294002 (10 µM, a PI3K/AKT pathway inhibitor; TargetMol) for 2 h, followed by rapamycin treatment.

Techniques: Inhibition, Over Expression, Quantitative RT-PCR, Immunofluorescence, Expressing, Control

Journal: Scientific Reports

Article Title: ERS regulates endometrial epithelial cell autophagy through XBP1s -mediated activation of the PI3K/AKT pathway

doi: 10.1038/s41598-024-84461-6

Figure Lengend Snippet: Effect of PI3K/AKT pathway activation by SC79 on knockdown of XBP1s. The experimental treatment conditions were as follows: pretreatment with SC79 for 2 h, followed by rapamycin treatment 24 h. (A-D) Relative protein expression levels of SQSTM1, ATG5, and the LC3II/LC3I were analyzed and determined using western blotting. (E-F) The relative mRNA expression levels of SQSTM1 and ATG5 analyzed and quantified using RT-qPCR after knockdown of XBP1s. Data are represented as the means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control group. # , P < 0.05; ## , P < 0.01; ### , P < 0.001 vs. other group.

Article Snippet: The medium was replaced with fresh medium and the following treatments were initiated: (1) rapamycin (50 nM; TargetMol, Boston, MA, USA) for 3 h, 6 h, 12 h, and 24 h; (2) pretreatment with TG (Thapsigargin; 50 nM; an ERS activator; TargetMol) or 4-PBA (4-Phenylbutyric acid; 1 mM, an ERS inhibitor; Abcam, Cambridge, UK) or SC79 (10 µM; a PI3K/AKT pathway activator; TargetMol) or LY294002 (10 µM, a PI3K/AKT pathway inhibitor; TargetMol) for 2 h, followed by rapamycin treatment.

Techniques: Activation Assay, Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: Cell reports

Article Title: Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins

doi: 10.1016/j.celrep.2018.04.009

Figure Lengend Snippet: (A) Expression pattern of Selenof and other relevant proteins across mouse tissues. (B-D) Expression of Selenof and other relevant proteins in MEFs from WT, heterozygous, and Selenof KO mice subjected to ER stressors thapsigargin (B), tunicamycin (C), and brefeldin A (D). WT, heterozygous, and KO MEFs were treated with two concentrations of stressors along with control (DMSO treated): thapsigargin (5 nM and 50 nM), tunicamycin (50 ng/mL and 500 ng/mL), and brefeldin A (0.5 μM and 5 mM). Proteins assayed are shown on the left.

Article Snippet: Tunicamycin, brefeldin A, and thapsigargin were from Tocris Bioscience (Bristol, UK); and lipopolysaccharide (LPS; L2637), SIGMAFAST OPD (o-phenylenediamine), and 2-mercaptoethanol were from Sigma (St. Louis, MO, USA).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

doi: 10.1101/2020.06.23.164731

Figure Lengend Snippet: A Reproduction of previously published data confirms loss of Bip-AMPylation upon ER stress by thapsigargin in WB. 20 µg treated (as indicated) ChoK1 cell lysate per lane or 50 ng recombinant Bip-AMP were analyzed in WB by antibody 17G6 and anti-Bip antibody. B Successful IP with antibody 17G6 on recombinant BiP-AMP confirms that antibody 17G6 is AMP-specific. C Successful IP of endogenous Bip-AMP with antibody 17G6 from treated (as indicated) ChoK1 cell lysates confirms applicability in immunoprecipitation. 50 ng recombinant Bip-AMP were blotted as control. D Using antibody 17G6 on various immortalized and cancer cell lines reveals diverse cellular AMPylation. 20 µg cell lysate per lane as indicated were blotted and probed with antibody 17G6 using 1 mM MnCl 2 as additive. Afterwards cells were treated with 1 M hydroxylamine to cleave ADP-ribosylation at aspartate and glutamate residues and reprobed with antibody 17G6 using 1 mM MnCl 2 . Antibodies against Bip, GAPDH and Histone H3 serve as loading control.

Article Snippet: 90% confluent cells were stimulated by either 0.5 μM thapsigargin (Biosynth Carbosynth) for 2 h or 100 μg/ml cycloheximide (Sigma-Aldrich) for 4 h. Cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich) and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with cOmplete EDTA free protease inhibitor (Roche, Basel, Switzerland).

Techniques: Recombinant, Immunoprecipitation, Control