Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: The multi-protective effect of IL-37-Smad3 against ox-LDL induced dysfunction of endothelial cells.

doi: 10.1016/j.biopha.2024.116268

Figure Lengend Snippet: Fig. 3. RH-IL37 (1 ng/mL) treatment suppressed ox-LDL induced endoplasmic reticulum stress expression in RCAECs dependent on Smad3 factor. The protein expression of IRE1-XBP1, PERK-eIF2α-ATF4, ATF6 pathway, and ERS markers CHOP, GRP78 in the cytoplasm of RCAECs assessed following exposed to ox-LDL (80 µg/mL) after pretreatment with or without RH-IL37 (1 ng/mL) or Smad3 silencing. The results demonstrated that ox-LDL increased the expression of IRE1- XBP1, PERK-eIF2α-ATF4, and ATF6 pathway components, as well as CHOP, GRP78 markers in RCAECs,. However, intervention with IL-37 significantly

Article Snippet: Furthermore, the antibodies against XBP1s (rabbit monoclonal antibody #4868–1-AP), ATF4 (rabbit monoclonal antibody #10835–1-AP), ALP (mouse monoclonal antibody #60294–1-Ig), RUNX2 (Rabbit monoclonal antibody #20700–1-AP), and BMP-2 (mouse monoclonal antibody #66383–1-Ig) were obtained from Proteintech (Wuhan Sanying, Wuhan, Hubei, P.R.China).

Techniques: Expressing

Journal: Biochemistry and Biophysics Reports

Article Title: Characterization of the 5′-flanking region of the human and mouse CHAC1 genes

doi: 10.1016/j.bbrep.2020.100834

Figure Lengend Snippet: CHAC1 is a UPR induced gene . Examinations of A) mRNA expressions of CHAC1, CHOP, C/EBPβ, GRP78 and control G3PDH in HEK293 cell extracts after treatments with UPR agonists. Treatments: Thapsigargin (Tg), Tunicamycin (Tm), Brefeldin A (BFA) or serum starvation (SF). B) Western blots to define protein expression of ATF4, CHOP and C/EBPβ or Acitin (loading control) in HEK293 cells after activation of the UPR using Tunicamycin treatment (Tm), versus control treated.

Article Snippet: Antibodies against ATF4 (sc-200), CHOP (sc-793), C/EBPβ (sc-150) were purchased from Santa Cruz Biotechnology and actin (CP01) was purchased from Calbiochem.

Techniques: Control, Western Blot, Expressing, Activation Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Characterization of the 5′-flanking region of the human and mouse CHAC1 genes

doi: 10.1016/j.bbrep.2020.100834

Figure Lengend Snippet: A) ATF4 activates the CHAC1 promoter acting synergistically at upstream ATF/CRE and CARE elements. Indicated derivatives of the CHAC1 promoter were linked to a luciferase reporter system for quantitative determination of transcriptional induction accompanying co-transfection of a mature ATF4 expressing plasmid or control. Comparison of ATF4 effects on CHAC1 transcription are also examined after mutations of the m1: ATF/CRE, m2: CARE, or m3: ATF/CRE + CARE. B) Synergistic actions of ATF4 on CHAC1 promoter ATF/CRE and CARE in mice and humans. Indicated derivatives of the CHAC1 promoter were linked to a luciferase reporter system for quantitative determination of transcriptional induction accompanying co-transfection of a mature ATF4 expressing plasmid or control. Comparison of ATF4 effects on CHAC1 transcription accompanying mutation of the m1: ATF/CRE, or m3: ATF/CRE + CARE. Values represent the mean ± SD from three independent cultures and are expressed relative to the basal activity of the pGL3-Basic vector. Data were analyzed by one way-ANOVA followed by Tukey's post test. The values marked with an asterisk highlight a statistical difference between the groups compared (p < 0.01).

Article Snippet: Antibodies against ATF4 (sc-200), CHOP (sc-793), C/EBPβ (sc-150) were purchased from Santa Cruz Biotechnology and actin (CP01) was purchased from Calbiochem.

Techniques: Luciferase, Cotransfection, Expressing, Plasmid Preparation, Control, Comparison, Mutagenesis, Activity Assay

Journal: Biochemistry and Biophysics Reports

Article Title: Characterization of the 5′-flanking region of the human and mouse CHAC1 genes

doi: 10.1016/j.bbrep.2020.100834

Figure Lengend Snippet: A) CHOP co-transfection inhibits expression of CHAC1, via the cis ATF/CRE element. Indicated derivatives of the CHAC1 promoter were linked to a luciferase reporter system for quantitative determination of transcriptional induction accompanying co-transfection of a mature CHOP expressing plasmid or control. Comparison of CHOP effects on CHAC1 transcription accompanying mutation of the m1: ATF/CRE, m2: CARE, or m3: ATF/CRE + CARE are examined. Values represent the mean ± SD from three independent cultures and are expressed relative to the basal activity of the pGL3-Basic vector. Data were analyzed by one way-ANOVA followed by Tukey's post test. The values marked with an asterisk highlight a statistical difference between the groups compared (p < 0.01). B ) Schematic of important cis elements of the CHAC1 promoter, and makeup of probes used for IM-EMSA to probe transcription factor binding to DNA sequences. C) ATF4 binds to the CHAC1 ATF/CRE after induction of the UPR . Examinations of ATF4 protein expression in native nuclear extracts after control (co) or induction of the UPR with 3 uM Thapsigargin for 24 h, and binding to probes corresponding to cis elements of the CHAC1 promoter. The respective probe added to each lane is listed at the top of the well. Slow mobility unbound ATF4 is shown at the top of the blot, and the presence of an increased mobility DNA-bound ATF4 is shown with an arrow. D) CHOP does not bind to the CHAC1 ATF/CRE, at baseline or after induction of the UPR. IM-EMSA examinations of CHOP protein expression in native nuclear extracts after control (co) or induction of the UPR with 3 uM Thapsigargin for 24 h, and binding to probes corresponding to cis elements of the CHAC1 promoter. The respective probe added to each lane is listed at the top of the well. A control CARE element from the TRIB3 gene (Trb3) is also examined. Slow mobility of free CHOP is shown at the top of the blot, and no indication of any increased mobility DNA-bound CHOP was detected in this assay.

Article Snippet: Antibodies against ATF4 (sc-200), CHOP (sc-793), C/EBPβ (sc-150) were purchased from Santa Cruz Biotechnology and actin (CP01) was purchased from Calbiochem.

Techniques: Cotransfection, Expressing, Luciferase, Plasmid Preparation, Control, Comparison, Mutagenesis, Activity Assay, Binding Assay

Journal: British Journal of Cancer

Article Title: KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression

doi: 10.1038/s41416-020-0872-0

Figure Lengend Snippet: a Ire1α and XBP1s expression in mock- and KSHV-infected macrophages was evaluated by western blot analysis; b ATF4, CHOP and BIP expression in mock- and KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of each protein/β-ACT. * p -value < 0.05. c PD-L1 expression on mock- and KSHV-infected macrophages was evaluated by FACS analysis. A representative experiment is shown, and the mean of fluorescence intensity is indicated. Grey peaks represent the isotype controls. d Histograms representing the mean plus SD of PD-L1 MFI (Mean fluorescence Intensity) are also reported. * p -value < 0.05.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against Kb-ZIP (Santa Cruz Biotechnology, sc-69797), pSTAT6 (1:100; Santa Cruz Biotechnology Inc., sc-136019), STAT6 (1:100; Santa Cruz Biotechnology Inc., sc-1689), mouse monoclonal anti-STAT3 (1:1000; BD Transduction Laboratories, 610189), mouse monoclonal anti-phospho-STAT3 (p-Tyr705, 1:100; Santa Cruz Biotechnology Inc., sc-8059), pSTAT1 (1:100; Santa Cruz Biotechnology Inc., sc-136229), STAT1 (1:100; Santa Cruz Biotechnology Inc., sc-464), mouse monoclonal anti-Ire1α (1:100; Santa Cruz Biotechnology, sc-390960), XBP1s (NovusBio NBP1-77681SS), ATF4 (R&D system, MAB7218), rabbit polyclonal anti-BIP (1:100; Cell Signaling, 3177), mouse monoclonal anti-CHOP (1:100; Santa Cruz Biotechnology, sc-7351), and anti-β-actin (1:10000; Sigma Aldrich, A5441).

Techniques: Expressing, Infection, Western Blot, Control, Fluorescence

Journal: British Journal of Cancer

Article Title: KSHV infection skews macrophage polarisation towards M2-like/TAM and activates Ire1 α-XBP1 axis up-regulating pro-tumorigenic cytokine release and PD-L1 expression

doi: 10.1038/s41416-020-0872-0

Figure Lengend Snippet: a IL-10, VEGF, IL-8, IL-6 and IFN-γ released by mock-, KSHV-infected macrophages and 4μ8c- (Ire1α inhibitor) or GSK 2606414 (GSK)- (PERK inhibitor) pre-treated KSHV-infected macrophages were measured by Luminex assay. Mean plus SD of three different experiments is reported. * p -value < 0.05; b and c PD-L1 expression on mock-, KSHV-infected macrophages and 4μ8c or GSK 2606414 (GSK)- pre-treated KSHV-infected macrophages was evaluated by FACS analysis. Grey peaks represent the isotype controls. Histograms representing the mean plus SD of PD-L1 MFI (Mean fluorescence Intensity) are reported. * p -value < 0.05 and a representative experiment is shown, and the mean of fluorescence intensity is indicated; d expression of K-bZIP in untreated or 4μ8c or GSK 2606414 (GSK)- pre-treated KSHV-infected macrophages; e ATF4 expression in mock-, KSHV-infected and GSK 2606414 (GSK)-pre-treated KSHV-infected macrophages was evaluated by western blot analysis. β-actin (β-ACT) was used as loading control. A representative experiment out of three is shown. Histograms represent the mean plus S.D. of the densitometric analysis of the ratio of each protein/ β-ACT. * p -value < 0.05.

Article Snippet: The following antibodies were used: mouse monoclonal antibody against Kb-ZIP (Santa Cruz Biotechnology, sc-69797), pSTAT6 (1:100; Santa Cruz Biotechnology Inc., sc-136019), STAT6 (1:100; Santa Cruz Biotechnology Inc., sc-1689), mouse monoclonal anti-STAT3 (1:1000; BD Transduction Laboratories, 610189), mouse monoclonal anti-phospho-STAT3 (p-Tyr705, 1:100; Santa Cruz Biotechnology Inc., sc-8059), pSTAT1 (1:100; Santa Cruz Biotechnology Inc., sc-136229), STAT1 (1:100; Santa Cruz Biotechnology Inc., sc-464), mouse monoclonal anti-Ire1α (1:100; Santa Cruz Biotechnology, sc-390960), XBP1s (NovusBio NBP1-77681SS), ATF4 (R&D system, MAB7218), rabbit polyclonal anti-BIP (1:100; Cell Signaling, 3177), mouse monoclonal anti-CHOP (1:100; Santa Cruz Biotechnology, sc-7351), and anti-β-actin (1:10000; Sigma Aldrich, A5441).

Techniques: Infection, Luminex, Expressing, Fluorescence, Western Blot, Control