primary antibodies against gpx4  (Proteintech)

Journal: International Journal of Biological Sciences

Article Title: Triptonide-mediated PTGS2 Inhibition Induces Autophagic Cell Death to Suppress the Progression of Triple-negative Breast Cancer and Epithelial Ovarian Cancer

doi: 10.7150/ijbs.127562

Figure Lengend Snippet: Triptonide targets the PTGS2 protein, which contributes to the progression of TNBC and EOC. (A) Representative images ( left ) and correlation analysis ( right ) of PTGS2 IHC score vs. Ki-67 index in TNBC tumor tissues ( top ), and PTGS2 IHC score vs. tumor volume in patients with EOC ( bottom ). (B) Western blot analysis of PTGS2 expression in shRNA control or PTGS2 -targeting shRNA-transduced SUM-159PT, MDA-MB-231. and SKOV3 cells. (C) CCK-8 assay of proliferation in shRNA control and PTGS2 -knockdown SUM-159PT, MDA-MB-231, and SKOV3 cells. (D) TN IC 50 curves for PTGS2 -knockdown and shRNA control SUM-159PT, MDA-MB-231, and SKOV3 cells. (E) TN IC 50 curves for vector control vs. PTGS2 -overexpressing SUM-159PT, MDA-MB-231, and SKOV3 cells. (F) Flow cytometry-evaluated apoptosis of PTGS2 -knockdown and shRNA control SUM-159PT, SKOV3 cells (20 nM TN, 48 h). (G) Assessment of apoptosis- and autophagy-related proteins via western blot in PTGS2-knockdown and shRNA control SUM-159PT, SKOV3 cells (40 nM TN, 48 h). Data are reported as mean ± SD, with statistical significance evaluated via Spearman's correlation analysis, two-way ANOVA, or Student's t-test; * P < 0.05, *** P < 0.001.

Article Snippet: For IHC analysis, tissue sections were subjected to antigen retrieval and blocking, then incubated overnight at 4°C with specific primary antibodies against PTGS2 (1:200 dilution, Cat# 12375-1-AP, Proteintech), Ki-67 (1:4000 dilution, Cat# 27309-1-AP, Proteintech), p62 (1:200 dilution, Cat# 18420-1-AP, Proteintech), c-Myc (1:500 dilution, Cat# 10828-1-AP, Proteintech), and pSTAT3 (1:100 dilution, Cat# 9145, Cell Signaling Technology).

Techniques: Western Blot, Expressing, shRNA, Control, CCK-8 Assay, Knockdown, Plasmid Preparation, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Triptonide-mediated PTGS2 Inhibition Induces Autophagic Cell Death to Suppress the Progression of Triple-negative Breast Cancer and Epithelial Ovarian Cancer

doi: 10.7150/ijbs.127562

Figure Lengend Snippet: Triptonide exerts anticancer bioactivity by directly binding to His-207 of the PTGS2 protein. (A) Molecular docking analysis of PTGS2 and TN. (B) Three-dimensional Gibbs free energy landscape of the PTGS2-TN binding system. (C) Analysis of the 100 ns molecular dynamics simulation trajectory of the PTGS2-TN complex. (D) Binding affinity between TN and PTGS2 protein measured by SPR analysis. (E-F) CETSA (E) and DARTs (F) assays were used to detect the binding between TN and PTGS2 in PTGS2-overexpressing HEK-293T cells. Representative blot ( left ) and quantitative analysis ( right ) are shown. (G) Free energy distribution at individual binding sites following TN binding to wild-type PTGS2. (H) Free energy distribution at binding sites after TN binding to PTGS2 Q203A and PTGS2 H207A mutants. (I) Schematic representation of wild-type PTGS2 and catalytic-site mutants. (J) Western blot analysis of PTGS2 expression in wild-type/mutant PTGS2 cell lines. (K) Viability of cells expressing wild-type or mutant PTGS2 after treatment with 40 nM TN or DMSO for 72 h. (L) CETSA analysis of TN binding to PTGS2 H207A mutant. Representative blot ( left ) and quantitative analysis ( right ) are shown. (M) Western blot analysis of degradation of PTGS2 H207A mutant protein in TN-treated cell lysate under pronase E treatment. Data are expressed as mean ± SD, with statistical significance evaluated via two-way ANOVA, or Student's t-test; * P < 0.05, ** P < 0.01, *** P < 0.001, ns, no significance.

Article Snippet: For IHC analysis, tissue sections were subjected to antigen retrieval and blocking, then incubated overnight at 4°C with specific primary antibodies against PTGS2 (1:200 dilution, Cat# 12375-1-AP, Proteintech), Ki-67 (1:4000 dilution, Cat# 27309-1-AP, Proteintech), p62 (1:200 dilution, Cat# 18420-1-AP, Proteintech), c-Myc (1:500 dilution, Cat# 10828-1-AP, Proteintech), and pSTAT3 (1:100 dilution, Cat# 9145, Cell Signaling Technology).

Techniques: Binding Assay, Western Blot, Expressing, Mutagenesis

Journal: International Journal of Biological Sciences

Article Title: Triptonide-mediated PTGS2 Inhibition Induces Autophagic Cell Death to Suppress the Progression of Triple-negative Breast Cancer and Epithelial Ovarian Cancer

doi: 10.7150/ijbs.127562

Figure Lengend Snippet: Triptonide promotes PTGS2 degradation via the ubiquitin-proteasome system by recruiting the E3 ligase NEDD4. (A) CETSA assay of TN-treated SUM-159PT cell lysate; DMSO-treated cell lysate was used as a negative control, and representative blot ( left ) and quantitative analysis ( right ) are shown. (B) PTGS2 protein expression in SUM-159PT, MDA-MB-231, and SKOV3 cells treated with 40 nM TN for 48 h was assessed by western blot. (C) The inhibitory effect of TN on PTGS2 protein expression was detected by western blot. (D) Effect of TN on PTGS2 expression in the presence of time-course CHX (100 μg/mL) treatment assessed by western blot. Representative blot ( left ) and quantitative analysis ( right ) are shown. (E) Effect of TN on PTGS2 expression following pretreatment with bafilomycin A1 (5 μM) or MG132 (10 μM), evaluated using western blot. Representative blot ( upper ) and quantitative analysis ( bottom ) are shown. (F) HEK-293T cells co-expressing PTGS2 and ubiquitin (or K48/K63 ubiquitin mutants) were pretreated with MG132 (10 μM, 6 h), followed TN (50 nM, 24 h) treatment, and the ubiquitination pattern of PTGS2 protein was assessed by co-IP assay. (G) Co-IP assay to examine the effect of TN on PTGS2 ubiquitination following K63 site mutation in ubiquitin. (H) HEK-293T cells co-expressing PTGS2 and NEDD4 were pretreated with MG132 and exposed to TN for 24 h. Co-IP assay was performed to detect the binding of PTGS2 and NEDD4. (I) CCK-8 detection of cell viability in TN-treated NEDD4-knockdown TNBC cells. Cell viability was measured using the CCK8 assay. (J) NEDD4-silenced SUM-159PT and MDA-MB-231 cells were subjected to 40 nM TN treatment for 48 h, with PTGS2 expression analyzed by western blot. Data are expressed as mean ± SD, with statistical significance assessed by two-way ANOVA, or Student's t-test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For IHC analysis, tissue sections were subjected to antigen retrieval and blocking, then incubated overnight at 4°C with specific primary antibodies against PTGS2 (1:200 dilution, Cat# 12375-1-AP, Proteintech), Ki-67 (1:4000 dilution, Cat# 27309-1-AP, Proteintech), p62 (1:200 dilution, Cat# 18420-1-AP, Proteintech), c-Myc (1:500 dilution, Cat# 10828-1-AP, Proteintech), and pSTAT3 (1:100 dilution, Cat# 9145, Cell Signaling Technology).

Techniques: Ubiquitin Proteomics, Negative Control, Expressing, Western Blot, Co-Immunoprecipitation Assay, Mutagenesis, Binding Assay, CCK-8 Assay, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Triptonide-mediated PTGS2 Inhibition Induces Autophagic Cell Death to Suppress the Progression of Triple-negative Breast Cancer and Epithelial Ovarian Cancer

doi: 10.7150/ijbs.127562

Figure Lengend Snippet: Triptonide induces autophagic cell death by downregulating the JAK/STAT3 signaling pathway. (A) Differential gene expression analysis (|log2FC| > 1, adjusted P < 0.05) in shRNA control and PTGS2 -knockdown SUM-159PT cells. (B) GSEA of downregulated genes in shRNA control and PTGS2 -knockdown SUM-159PT cells. (C-D) Western blot analysis of KRAS/ERK signaling (C) and STAT3/c-Myc signaling (D) in SUM-159PT, MDA-MB-231, and SKOV3 cells after TN (40 nM, 72 h) treatment. (E) MYC mRNA quantification by qPCR in TN-exposed SUM-159PT, MDA-MB-231, and SKOV3 cells. (F) GSEA of HALLMARK_IL6_JAK_STAT3_ SIGNALING and KEGG_JAK_STAT_SIGNALING pathways in shRNA control and PTGS2 -knockdown SUM-159PT cells. (G) After TN pretreatment (40 nM, 12 h), SUM-159PT, MDA-MB-231, and SKOV3 cells were exposed to TG101209 (1 or 2 µM, 24 h), and viability was measured using the CCK-8 method. (H) Cytotoxic effect of TN in SUM-159PT, MDA-MB-231, and SKOV3 cells after addition of ML115 (10 µM). (I) Changes in p62 and LC3-II protein levels in SUM-159PT and SKOV3 cells after TN and ML115 treatment, detected by western blot. (J) Apoptosis in SUM-159PT and SKOV3 cells after TN and ML115 treatment, assessed by flow cytometry. Data are expressed as mean ± SD, with statistical significance assessed by two-way ANOVA, or Student's t-test; * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: For IHC analysis, tissue sections were subjected to antigen retrieval and blocking, then incubated overnight at 4°C with specific primary antibodies against PTGS2 (1:200 dilution, Cat# 12375-1-AP, Proteintech), Ki-67 (1:4000 dilution, Cat# 27309-1-AP, Proteintech), p62 (1:200 dilution, Cat# 18420-1-AP, Proteintech), c-Myc (1:500 dilution, Cat# 10828-1-AP, Proteintech), and pSTAT3 (1:100 dilution, Cat# 9145, Cell Signaling Technology).

Techniques: Gene Expression, shRNA, Control, Knockdown, Western Blot, Protein-Protein interactions, CCK-8 Assay, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: Triptonide-mediated PTGS2 Inhibition Induces Autophagic Cell Death to Suppress the Progression of Triple-negative Breast Cancer and Epithelial Ovarian Cancer

doi: 10.7150/ijbs.127562

Figure Lengend Snippet: Triptonide suppresses the growth of TNBC and EOC tumors in vivo . (A-C) MDA-MB-231-derived xenograft mice divided into three groups (n = 6/group) were administered either the vehicle control, TN treatment, or positive control treatment. The resultant treatment effects were evaluated by tumor photographs (A), xenograft growth curves (B), and final tumor weights (C). (D-F) Three groups of SKOV3-derived xenograft mice (n = 6/group) were established and treated with vehicle control, TN, or positive control, respectively. The resultant treatment effects were evaluated by tumor photographs (D), xenograft growth curves (E), and final tumor weights (F). (G-J) IHC staining of Ki-67 (G), p62 (H), PTGS2 (I), and pSTAT3 (J) expression in vehicle-treated and TN-treated groups. Representative images ( left ) and quantitative analysis ( right ) are shown. Data are expressed as mean ± SD, with statistical significance assessed by one-way ANOVA, or Student's t-test; ** P < 0.01, *** P < 0.001.

Article Snippet: For IHC analysis, tissue sections were subjected to antigen retrieval and blocking, then incubated overnight at 4°C with specific primary antibodies against PTGS2 (1:200 dilution, Cat# 12375-1-AP, Proteintech), Ki-67 (1:4000 dilution, Cat# 27309-1-AP, Proteintech), p62 (1:200 dilution, Cat# 18420-1-AP, Proteintech), c-Myc (1:500 dilution, Cat# 10828-1-AP, Proteintech), and pSTAT3 (1:100 dilution, Cat# 9145, Cell Signaling Technology).

Techniques: In Vivo, Derivative Assay, Control, Positive Control, Immunohistochemistry, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin Protects Myocardium From Ischaemia/Reperfusion Injury by Inhibiting Ferroptosis Through Downregulation of VDAC1

doi: 10.1111/jcmm.70313

Figure Lengend Snippet: PUE pretreatment attenuates myocardial ferroptosis after I/R injury in mice by downregulating VDAC1. (A) Western blot detection of PTGS2 protein, GPX4 protein and VDAC1 protein expression in mouse myocardial lysates. (B, C, D) Histogram of PTGS2 protein, GPX4 protein and VDAC1 protein expression. (E) MDA level in mouse myocardial lysates. (F) Total iron level in mouse myocardial lysates. (G) GSH and GSSG ratios in mouse myocardial lysates. (H) The image of the myocardium was stained with DHE (magnification, ×400). Data are expressed as the mean ± SD ( n = 3 or 6). ns, nonsignificant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies against VDAC1 and PTGS2 were obtained from Proteintech (Chicago, IL, USA).

Techniques: Western Blot, Expressing, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin Protects Myocardium From Ischaemia/Reperfusion Injury by Inhibiting Ferroptosis Through Downregulation of VDAC1

doi: 10.1111/jcmm.70313

Figure Lengend Snippet: PUE inhibits ferroptosis of H/R‐induced H9c2 cells by mediating VDAC1. (A) Histogram of CCK‐8 detected the cell viability in H/R‐induced cells after treatment of each group separately. (B) Histogram of LDH activity in H/R‐induced cells after treatment of each group separately. (C) Western blot detection of PTGS2 protein, GPX4 protein and VDAC1 protein expression in H/R‐induced cells after treatment of each group separately. (D, E, F) Histogram of PTGS2 protein, GPX4 protein and VDAC1 protein expression. (G) Transmission electron microscopy images of H9c2 cells (magnification, ×6,000; scale bar, 1 μm). (H) Histogram of Flameng scores. (I) Total iron level in H/R‐induced cells after treatment of each group separately. (J) MDA level in H/R‐induced cells after treatment of each group separately. (K) 4‐HNE level after treatment of each group separately. Data are expressed as the mean ± SD ( n = 3). ns, nonsignificant, * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Primary antibodies against VDAC1 and PTGS2 were obtained from Proteintech (Chicago, IL, USA).

Techniques: CCK-8 Assay, Activity Assay, Western Blot, Expressing, Transmission Assay, Electron Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin Protects Myocardium From Ischaemia/Reperfusion Injury by Inhibiting Ferroptosis Through Downregulation of VDAC1

doi: 10.1111/jcmm.70313

Figure Lengend Snippet: PUE inhibits ferrous iron generation and lipid oxidation in H/R‐induced H9c2 cells by mediating VDAC1. (A) Intracellular Fe 2+ assessed by FerroOrange in H/R‐induced H9c2 cells after treatment of each group separately (magnification, ×200; scale bar, 400 μm). (B) Representative images of C11‐BODIPY (581/591) in H/R‐induced H9c2 cells after treatment of each group separately (magnification, ×200; scale bar, 400 μm). Data are expressed as the mean ± SD ( n = 3).

Article Snippet: Primary antibodies against VDAC1 and PTGS2 were obtained from Proteintech (Chicago, IL, USA).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Puerarin Protects Myocardium From Ischaemia/Reperfusion Injury by Inhibiting Ferroptosis Through Downregulation of VDAC1

doi: 10.1111/jcmm.70313

Figure Lengend Snippet: Schematic diagram of the mechanism by which PUE protects myocardium from I/R injury through VDAC1.

Article Snippet: Primary antibodies against VDAC1 and PTGS2 were obtained from Proteintech (Chicago, IL, USA).

Techniques:

Journal: Heliyon

Article Title: Dexmedetomidine preconditioning attenuates ferroptosis in myocardial ischemia-reperfusion injury via α2 adrenergic receptor activation.

doi: 10.1016/j.heliyon.2024.e39697

Figure Lengend Snippet: Fig. 5. Dex preconditioning enhanced antioxidant activity and reduced inflammation in rat myocardial tissues. (A) Nrf2 protein level. (B) COX2 protein level. (C) IL-1β mRNA expression. (D) IL-6 mRNA expression. (E) TNF-α mRNA expression. n = 3/group. All data are presented as the mean ± SEM. *P < 0.05 vs. the Sham group. #P < 0.05 vs. the I/R group. &P < 0.05 vs. the Dex + I/R group.

Article Snippet: Primary antibodies against COX2 (Cat. ab179800), transferrin receptor 1 (TFR1, Cat. ab269513), Ferritin (Cat. ab75973), ASCL4 (Cat. ab155282), SLC7A11 (Cat. ab175186), GPX4 (Cat. ab125066) (all from Abcam), and Nrf2 (Cat. 16396-1-AP, Proteintech) were incubated overnight at 4 ◦C.

Techniques: Antioxidant Activity Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Effects of blastocyst elongation and implantation chamber formation on the alignment of the embryonic axis and uterine axis in mice

doi: 10.3389/fcell.2024.1421222

Figure Lengend Snippet: Ptgs2 expression in the luminal epithelium at E4.0. (A) Immunofluorescence detection of Ptgs2 in the blastocyst in the E4.0 uterus. Ptgs2 was expressed in the LE attached to the mural and lateral regions of the blastocyst. Blue, green, and magenta signals represent Hoechst, Ptgs2, and F-actin, respectively. (B) Neighboring section of (A) . Stained by Hoechst and E-cadherin. Blue and green signal represent Hoechst and E-cadherin, respectively.

Article Snippet: Primary antibody against Ptgs2 was purchased (Cayman Chemical Cat# 160126, RRID:AB_10079419, 1:200).

Techniques: Expressing, Immunofluorescence, Staining