ptges3  (Proteintech)

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: Overexpression of PTGES3 in HCC and its association with patient prognosis. a Up-regulation of PTGES3 mRNA in public HCC datasets. Raincloud plots showing log 2 -transformed expression levels in five independent paired cohorts (sample sizes as indicated). Gray lines connect matched normal and tumor tissues. Box plots denote median and interquartile range (IQR); violin plots show distribution density. b , c, Validation of PTGES3 overexpression in clinical samples. b Representative immunoblots showing PTGES3 protein levels in paired tumor (T) and adjacent normal (N) tissues ( n = 3 biologically independent pairs). β-actin served as a loading control. c Densitometric quantification of protein abundance (left) and qPCR analysis of PTGES3 mRNA (right). Tumor expression levels are presented as fold change relative to normal counterparts. Data are presented as relative expression levels normalized to adjacent normal tissues. d Immunohistochemical (IHC) validation in tissue microarrays. Representative staining of PTGES3 in paired normal and tumor tissues ( n = 87 pairs). Scale bars, 200 μm (cores) and 50 μm (insets). Right, paired quantification of IHC scores (arbitrary units, a.u.). Box plots indicate median and IQR; gray lines connect matched samples. e , f , Association with poor clinical outcomes. Kaplan–Meier curves for disease-free survival (DFS; e ) and overall survival (OS; f ) in the institutional cohort ( n = 87). Patients were stratified into high (red) and low (blue) expression groups based on median PTGES3 IHC scores. Shaded areas, 95% confidence interval (CI); crosses, censored data. Statistical analysis. Data are presented as mean ± SEM ( c ) or median with interquartile range ( a, d ). Statistical significance was determined using the Wilcoxon signed-rank test ( a ), paired t -test ( d ), Two-way ANOVA with Šídák's post hoc test ( c ), or log-rank test ( e , f ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: HCC, hepatocellular carcinoma; GEO, Gene Expression Omnibus; ICGC, International Cancer Genome Consortium; NODE, National Omics Data Encyclopedia; IHC, immunohistochemistry; qPCR, quantitative polymerase chain reaction; IQR, interquartile range; DFS, disease-free survival; OS, overall survival; T, tumor; N, normal; a.u., arbitrary units; Rel., relative; SEM, standard error of the mean; ANOVA, analysis of variance; CI, confidence interval

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: Over Expression, Transformation Assay, Expressing, Biomarker Discovery, Western Blot, Control, Quantitative Proteomics, Immunohistochemical staining, Staining, Gene Expression, Immunohistochemistry, Real-time Polymerase Chain Reaction

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: PTGES3 promotes HCC cell proliferation, migration, and survival in Huh7 cells. a Cell viability assays. MTT assays in Huh7 cells following PTGES3 knockdown (screening of three siRNAs, left) or overexpression (right) at 48 h ( n = 3 independent biological replicates; n = 8 technical replicates for each). Data are normalized to respective controls (siNC for knockdown; Vector for overexpression). b Colony formation assays. Representative images (left) and quantification (right) of colonies formed by Huh7 cells following PTGES3 knockdown (using shNC/sh PTGES3 ) or overexpression ( n = 3 independent biological replicates). c Migration assays. Representative images of wound closure at 0 and 48 h (left) and quantification of migration rates (right) ( n = 3 independent biological replicates). Scale bars, 100 μm. d Apoptosis analysis. Representative flow cytometry plots (left) and quantification (right) in Huh7 cells following PTGES3 knockdown (siNC vs. si PTGES3 -1) ( n = 4 independent biological replicates). The apoptotic rate represents the combined percentage of early (Annexin V⁺/7-AAD⁻) and late (Annexin V⁺/7-AAD⁺) apoptotic cells. Statistical analysis. Data are presented as mean ± SEM. Individual values are superimposed on bars. Significance was determined using One-way ANOVA with Dunnett's post hoc test (for siRNA screening in a), Tukey's post hoc test (for b, c), or two-tailed unpaired Student's t -test (for Vector vs. PTGES3 in a, c and siNC vs. si PTGES3 in d). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: HCC, hepatocellular carcinoma; MTT, 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; shRNA, short hairpin RNA; 7-AAD, 7-aminoactinomycin D; Rel., relative; SEM, standard error of the mean; ANOVA, analysis of variance; NC, negative control

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: Migration, Knockdown, Over Expression, Plasmid Preparation, Flow Cytometry, Two Tailed Test, Small Interfering RNA, shRNA, Negative Control

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: PTGES3 activates the PI3K/AKT/mTOR signaling pathway to promote HCC progression. a Volcano plot. Analysis of differentially expressed genes (DEGs) in Huh7 cells following PTGES3 knockdown ( n = 3; |log₂FC|> 0.5, adjusted P < 0.05). Red dots (124 genes) and blue dots (181 genes) indicate upregulated and downregulated genes, respectively. b KEGG pathway enrichment analysis. Bubble chart showing the top enriched signaling pathways, identifying the PI3K/AKT pathway as a primary downstream target. c Western blot validation. Immunoblotting of key PI3K/AKT/mTOR pathway components (PI3K, p-AKT, t-AKT, p-mTOR, t-mTOR, p-P70S6K, t-P70S6K, p-4EBP1, and t-4EBP1) in Huh7 cells following PTGES3 knockdown (using shNC/sh PTGES3 ) or overexpression (Vector/ PTGES3 ) ( n = 3 independent biological experiments). β-actin served as the loading control. d Cell viability rescue. MTT assays in PTGES3 -overexpressing Huh7 cells treated with or without Rapamycin (100 nM) at 48 h ( n = 3 independent biological replicates; n = 8 technical replicates for each). Data are presented as relative cell viability normalized to the untreated control group. e Migration rescue. Representative images of wound closure (0 h vs. 48 h) and quantitative analysis of migration rates in Huh7 cells ( n = 3 independent biological replicates). Scale bars, 100 μm. f Colony formation rescue. Quantitative analysis of colonies formed by Huh7 cells showing that Rapamycin suppresses PTGES3-induced growth enhancement ( n = 3 independent biological replicates). Statistical analysis. Data are presented as mean ± SEM. Individual data points for all biological replicates are superimposed on the bars. Statistical significance was determined using Wald test with Benjamini–Hochberg correction (for transcriptomic analysis in a) or Two-way ANOVA with Tukey's post hoc test (for multi-group comparisons in d–f). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: HCC, hepatocellular carcinoma; DEGs, differentially expressed genes; FC, fold change; KEGG, Kyoto Encyclopedia of Genes and Genomes; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; P70S6K, ribosomal protein S6 kinase beta-1; 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; p-, phosphorylated; t-, total; MTT, 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide; shRNA, short hairpin RNA; Rel., relative; SEM, standard error of the mean; ANOVA, analysis of variance; NC, negative control

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: Knockdown, Protein-Protein interactions, Western Blot, Biomarker Discovery, Over Expression, Plasmid Preparation, Control, Migration, Binding Assay, shRNA, Negative Control

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: Ptges3 silencing suppresses DEN-induced hepatocarcinogenesis in vivo. a Experimental schema. Timeline of DEN induction and hepatocyte-specific AAV intervention in 2-week-old male mice ( n = 15 per group). b Micro-CT assessment. (Left) Representative scans across transverse, coronal, and sagittal planes; red solid triangles indicate the measured internal lesions. Two typical images per group are displayed. (Right) Quantification of the maximum tumor diameter per mouse at week 22 (shNC, n = 12; sh Ptges3 , n = 10). c Macroscopic observation. Representative photographs of livers at week 24. Red solid triangles indicate tumor nodules on the liver surface. d Histopathology. Representative H&E staining showing shNC and sh Ptges3 group tumor tissue. Scale bars, 200 μm (top), 50 μm (bottom). e , f , IHC analysis. Representative IHC images and quantification of PTGES3 ( e ) and Ki-67 ( f ) expression in HCC sections at week 24 ( n = 6 independent mice per group). Scale bars, 200 μm (left), 50 μm (right). Statistical analysis. Data are presented as mean ± SEM. Individual values for each mouse are superimposed on the bars. Statistical significance was determined using Welch's t -test (for b) or two-tailed unpaired Student's t -test (for e, f). ** P < 0.01, *** P < 0.001. Abbreviations: HCC, hepatocellular carcinoma; DEN, diethylnitrosamine; AAV, adeno-associated virus; shRNA, short hairpin RNA; CT, computed tomography; H&E, hematoxylin and eosin; T, tumor; N, non-tumor; IHC, immunohistochemistry; OD, optical density; TBG, thyroxine-binding globulin; SEM, standard error of the mean; NC, negative control

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: In Vivo, Micro-CT, Histopathology, Staining, Expressing, Two Tailed Test, Virus, shRNA, Computed Tomography, Immunohistochemistry, Binding Assay, Negative Control

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: PTGES3 drives M2 macrophage polarization in the HCC microenvironment via the TGF-β axis. a Global scRNA-seq landscape. t-SNE visualization of integrated cell clusters identified from shNC and sh Ptges3 mouse hepatic tissues ( n = 3 independent biological replicates), identifying nine major cell types. b Cellular composition. Relative cell fractions of intrahepatic immune populations showing the reduction of the macrophage population in the sh Ptges3 group. c Polarization signatures. Violin plots comparing M1-type scores, M2-type scores, and M1/M2 polarization ratios in macrophages based on scRNA-seq transcriptomic data. d In vivo validation. Representative IF images and mean fluorescence intensity quantification (a.u.) of CD206 (red) and DAPI (blue) in mouse HCC tissues ( n = 6 mice per group). Scale bars, 20 μm. e FACS analysis. Representative histograms showing the percentage of CD14⁺CD163⁺ M2 macrophages (left) and CD14⁺CD86⁺ M1 macrophages (right) in the Huh7/M0 co-culture system following PTGES3 knockdown (quantification in Fig. S4a). f PGE2 assessment. ELISA quantification of PGE2 in Huh7 supernatants following PTGES3 knockdown (si PTGES3 ) or overexpression (Vector/ PTGES3 ) ( n = 6 independent biological replicates). g Cytokine mRNA profiling. Bar charts showing the average expression levels of M2-inducing cytokines ( Il4 , Il10 , Il13 , Tgfb1 ) in macrophages from the scRNA-seq dataset. h , i , TGF-β quantification. ELISA measurement of TGF-β concentrations in mouse hepatic tissues ( h , n = 4 independent biological replicates) and Huh7 cell supernatants ( i , n = 3 independent biological replicates). j Rescue assay. Representative Western blot images and quantitative analysis of the M2 marker CD163 in macrophages co-cultured with PTGES3 -silenced Huh7 cells in the presence or absence of exogenous TGF-β (10 ng/mL). β-actin served as the loading control. Bar graphs display the relative protein expression levels normalized to the M0 group ( n = 3 independent biological replicates). Statistical analysis. Data are presented as mean ± SEM. Individual data points are superimposed on the bars. Statistical significance was determined using Wilcoxon rank-sum test (for scRNA-seq analysis in b , c ), Wilcoxon signed-rank test (for g ), two-tailed unpaired Student's t -test (for comparisons in d , f , h , i ), or One-way ANOVA with Tukey's post hoc test (for rescue assays in j ). * P < 0.05, *** P < 0.001, **** P < 0.0001; ns, not significant. Abbreviations: HCC, hepatocellular carcinoma; scRNA-seq, single-cell RNA sequencing; t-SNE, t-distributed stochastic neighbor embedding; IF, immunofluorescence; DAPI, 4',6-diamidino-2-phenylindole; Mφ, macrophage; co-cul., co-culture; FACS, fluorescence-activated cell sorting; ELISA, enzyme-linked immunosorbent assay; PGE2, prostaglandin E2; TGF-β, transforming growth factor-beta; shRNA, short hairpin RNA; a.u., arbitrary units; Rel., relative; SEM, standard error of the mean; ANOVA, analysis of variance; NC, negative control

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: In Vivo, Biomarker Discovery, Fluorescence, Co-Culture Assay, Knockdown, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Expressing, Rescue Assay, Western Blot, Marker, Cell Culture, Control, Two Tailed Test, Single Cell, RNA Sequencing, Immunofluorescence, FACS, shRNA, Negative Control

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: Nuclear PTGES3 directly binds and transcriptionally activates SP1 . a Target identification. Venn diagram displaying the intersection between PTGES3-bound genes (identified by CUT&Tag) and known transcriptional regulators of TGFB1 (retrieved from the TRRUST v2 database). b Peak visualization. Representative Integrative Genomics Viewer (IGV) tracks showing specific PTGES3 enrichment at the SP1 promoter region. c Motif analysis. The specific G-rich motif sequence identified within the PTGES3 binding peak. d Direct binding verification (EMSA). Electrophoretic mobility shift assay with biotinylated SP1 promoter probes and purified PTGES3 protein. Lane 1: Free probe; Lane 2: Probe + Protein (Shift); Lane 3: Probe + Protein + 5 × WT Competitor (Competition); Lane 4: Probe + Protein + 5 × Mutant Competitor (No competition). e ChIP-qPCR. Chromatin immunoprecipitation validating PTGES3 recruitment to the SP1 promoter in Huh7 cells ( n = 3). Cells expressing negative control shRNA (shNC) or PTGES3 -targeting shRNA (sh PTGES3 ) were assayed using non-specific immunoglobulin G (IgG, black bars) or specific anti-PTGES3 antibodies (red bars). f Transcriptional activity. Dual-luciferase reporter assay in Huh7 cells ( n = 3). Cells were co-transfected with reporter plasmids containing either the Wild-Type (WT) or motif-mutated (Mut) SP1 promoter, alongside empty vector (Vector, white bars) or PTGES3 expression plasmids (red bars). Motif mutation completely abolishes PTGES3-mediated activation. g , mRNA regulation. qPCR analysis of SP1 mRNA levels in Huh7 cells ( n = 3). Comparisons are between shNC and sh PTGES3 groups. h Protein regulation. Western blot analysis and quantification of SP1 expression in Huh7 cells ( n = 3). Left panels: knockdown (shNC vs. sh PTGES3 ); Right panels: overexpression (Vector vs. PTGES3 ). i TGF-β secretion. ELISA quantification of TGF-β levels in the supernatant of Huh7 cells ( n = 4). Groups: siNC + Vector (control), si SP1 + Vector ( SP1 knockdown alone), siNC + PTGES3 ( PTGES3 overexpression), and si SP1 + PTGES3 (rescue). j Signaling rescue (si SP1 ). Western blot analysis of PI3K/AKT/mTOR pathway components in Huh7 cells. SP1 silencing (si SP1 + PTGES3 ) reverses pathway activation induced by PTGES3 alone (siNC + PTGES3 ). k Receptor dependency (ITD-1). Western blot analysis of the PI3K/AKT pathway in Huh7 cells treated with the selective TGF-β receptor inhibitor ITD-1 (5 μM). Groups: Vector vs. PTGES3 overexpression in the presence or absence of ITD-1. l HSP90 independence (17-AAG). Western blot analysis in Huh7 cells treated with the HSP90 inhibitor 17-AAG (0.5 μM). Statistical analysis. Data are presented as mean ± SEM. Individual values are superimposed on bars. Significance was determined using two-tailed unpaired Student's t -test (for g, h), Two-way ANOVA with Šídák's post hoc test (for e, f, i). ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. Abbreviations: CUT&Tag, Cleavage Under Targets and Tagmentation; IGV, Integrative Genomics Viewer; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; qPCR, quantitative polymerase chain reaction; shRNA, short hairpin RNA; IgG, immunoglobulin G; siRNA, small interfering RNA; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; Mut, mutant; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; TGF-β, transforming growth factor-beta; HSP90, heat shock protein 90; ANOVA, analysis of variance; SEM, standard error of the mean; NC, negative control

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: Drug discovery, Sequencing, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Mutagenesis, ChIP-qPCR, Chromatin Immunoprecipitation, Expressing, Negative Control, shRNA, Activity Assay, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activation Assay, Western Blot, Knockdown, Over Expression, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Molecular Biomedicine

Article Title: Nuclear prostaglandin E synthase 3 promotes hepatocellular carcinoma growth with immunosuppressive macrophage polarization via the SP1/TGF-β axis

doi: 10.1186/s43556-026-00431-6

Figure Lengend Snippet: Schematic of the nuclear PTGES3/SP1/TGF-β signaling axis. The model illustrates that PTGES3 translocates to the nucleus and binds specifically to the G-rich motif of the SP1 promoter, driving SP1 transcription. Upregulated SP1 promotes TGF-β secretion, which drives HCC progression via two parallel signaling loops: (1) Autocrine loop: Secreted TGF-β binds to TGFBR on HCC cells, triggering the phosphorylation (P) of the PI3K/AKT/mTOR cascade to sustain tumor proliferation and migration. This axis was functionally validated using the specific inhibitors ITD-1 (targeting TGFBR) and Rapamycin (targeting mTOR), as indicated by the inhibitory symbols (⊥); (2) Paracrine loop: Secreted TGF-β acts on tumor-associated macrophages (TAMs) to induce CD206⁺ M2 polarization, establishing an immunosuppressive microenvironment. Abbreviations: HCC, hepatocellular carcinoma; TGF-β, transforming growth factor-beta; TGFBR, TGF-β receptor; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; 4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; TAM, tumor-associated macrophage; M2, alternatively activated macrophage; P, phosphorylation

Article Snippet: Cells were incubated with anti-PTGES3 antibody (CST #13,926) or IgG.

Techniques: Phospho-proteomics, Migration, Binding Assay