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: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Determination of MIF, COX2, and PGE2 expression in the posterior joint capsule after PTJC. A , Experimental design and flowchart of the animal experiment. B , Western blot analysis of MIF protein levels in posterior joint capsule following PTJC at 0, 1, 3, and 7 days. C, Quantitative results of B. D , Western blot analysis of COX1 and COX2 protein levels in posterior joint capsule following PTJC at 0, 1, 3, and 7 days. E and F, Quantitative results of D. G , PGE2 production was measured using ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * p < 0.05 compared with the 0 d group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Effects of MIF inhibitor 4-IPP on the COX2 expression and PGE2 production in vivo . A , Western blot analysis of COX2 protein levels in posterior joint capsule treated with or without 10 µL 4-IPP (100 mM) at 0, 1, 3, and 7 days post-injury. B, Quantitative results of A. C, Immunostaining of COX-2 (red) in posterior joint capsule treated with or without 4-IPP at 0 day and 3 days post-injury. Vimentin was used as fibroblast marker (green). Cell nucleus were stained with DAPI (blue). Scale bar, 20 µm. D , PGE2 production in posterior joint capsule was measured using ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with 0 d group. # P < 0.05 compared with Vehicle group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Expressing, In Vivo, Western Blot, Immunostaining, Marker, Staining, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Determination of COX2 expression and PGE2 production in response to MIF stimulation in joint capsule fibroblasts. A , Primary joint capsule fibroblasts were cultured and verified by classical microscopy image (BF), immunofluorescence, and immunohistochemical staining. Vimentin was used as fibroblast marker (green). Cell nucleus were stained with DAPI (blue). The purity of the isolated fibroblasts exceeded 95%. Scale bar, 100 μm. B-D , COX2 expression were assessed by qRT-PCR ( B ) and western blot ( C ) following joint capsule fibroblasts treatment with 0–2.5 μg/mL MIF for 24 h. Quantitative results of C as shown in ( D ). E , PGE2 production in supernatant and lysate of joint capsule fibroblasts were tested by ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with 0 μg/mL group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Expressing, Cell Culture, Microscopy, Immunofluorescence, Immunohistochemical staining, Staining, Marker, Isolation, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Effects of MIF inhibitor 4-IPP on the COX2 expression and PGE2 production in vitro . A-D, COX2 expression in joint capsule fibroblasts after treatment with 2 µg/mL MIF combined with 50 µM 4-IPP for 24 h was determined by qRT-PCR ( A ), western blot ( B ), and immunofluorescence ( D , Phalloidin was used as cytoskeleton (red) ) . Scale bar, 100 µm. Quantitative results of B as shown in ( C ). E , PGE2 production in supernatant and lysate of joint capsule fibroblasts were tested by ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with Control group or 4-IPP group. # P < 0.05 compared with MIF group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Expressing, In Vitro, Quantitative RT-PCR, Western Blot, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Effects of CD74 receptor and MAPK pathway on the MIF-induced COX2 expression and PGE2 production. A , Colocalization of CD74 (green) and MIF (red) in posterior joint capsule at 3 days post-injury. Scale bar, 20 µm. B-E , Joint capsule fibroblasts were treated with siRNA2 or control siRNA for 48 h and then treated with 2 µg/mL MIF for 24 h. qRT-PCR analysis of COX2 expression ( B ). Western blot ( C ) analysis of CD74 and COX2 expression. Quantitative results of C as shown in ( D ) and ( E ). F , PGE2 production in supernatant and lysate of joint capsule fibroblasts were tested by ELISA accordingly. G , Western blot analysis of COX2 expression following joint capsule fibroblasts were pretreated with 10 μM inhibitor of P38 (SB203580), JNK (SP600125), or ERK (PD98059) for 1 h before treatment with 2 μg/mL MIF for 24 h. H , Quantitative results of G. I and J , PGE2 production in supernatant ( I ) and lysate ( J ) of joint capsule fibroblasts were tested by ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with Control group. # P < 0.05 compared with MIF group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Effects of CREB on the MIF-induced COX2 expression and PGE2 production. A , Western blot analysis of p-CREB following joint capsule fibroblasts treated with 2 µg/mL MIF for 0, 15, 30, 60, and 120 min. B , Quantitative results of A. C , Western blot analysis of p-CREB in joint capsule fibroblasts following siRNA2 knockdown of CD74 for 48 h and 2 µg/mL MIF stimulation for 15 min. D , Quantitative results of C. E , Evaluation of CREB knockdown efficiency using qRT-PCR after transfection for 48 h and siRNA1 was selected for subsequent experiments. F–H , Following siRNA1 knockdown of CREB for 48 h and 2 μg/ml MIF stimulation for 24 h, COX2 expression in joint capsule fibroblasts was assessed by qRT-PCR ( F ) and Western blot ( G ). Quantitative results of G as shown in ( H ). I and J , PGE2 production in supernatant ( I ) and lysate ( J ) of joint capsule fibroblasts were tested by ELISA accordingly. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with Control group or 0 min group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Expressing, Western Blot, Knockdown, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Macrophage migration inhibitory factor mediates joint capsule fibrosis via facilitating phospholipid metabolite PGE2 production in fibroblasts

doi: 10.1007/s00018-025-05800-y

Figure Lengend Snippet: Effects of COX2 inhibitor NS398 on the proinflammatory cytokine production and polarization of macrophages. A - F , ELISA was performed to evaluate IL-1β ( A and B ), IL-6 ( C and D ), or TNF-α ( E and F ) production in both supernatant and lysate of macrophages RAW 264.7 following treatment with fibroblasts-conditioned medium in the presence of 1 µg/mL LPS. The fibroblasts-conditioned medium was prepared by joint capsule fibroblasts treatment with 2 μg/mL MIF with or without 30 μM NS398 for 24 h. G-J , Arginase 1 ( G ), Il10 ( H ), Nos2 ( I ), and Il12 ( J ) expression in macrophages RAW 264.7 was assessed by qRT-PCR after treatment with fibroblasts-conditioned medium for 24 h. The fibroblasts-conditioned medium was prepared by joint capsule fibroblasts treatment with 2 μg/mL MIF with or without 30 μM NS398 for 24 h. Each experiment was conducted three times. Error bars denote standard deviation. * P < 0.05 compared with fibroblasts group. # P < 0.05 compared with fibroblasts + MIF group

Article Snippet: Subsequently, joint capsule fibroblasts incubated with primary antibodies against COX2 (Cayman, 1:100), phalloidin (Abcam, 1:1000), and vimentin (Abcam, 1:1000) overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Standard Deviation