Journal: Journal of translational medicine

Article Title: An integrated approach of network pharmacology, molecular docking, and experimental verification uncovers kaempferol as the effective modulator of HSD17B1 for treatment of endometrial cancer.

doi: 10.1186/s12967-023-04048-z

Figure Lengend Snippet: Fig. 5 Kaempferol modulated estrogen metabolism pathways and differentially regulates PPARG expression in EC cells of different ER subtypes. A– B HSD17B1 and HSD17B1-associated genes, such as ESRRA, PPARG, and ESR1, are involved in several estrogen metabolism pathways, such as steroid binding, 17- beta-hydroxysteroid dehydrogenase (NADP+) activity, steroid hormone biosynthesis, and regulation of hormone levels. C Kaempferol suppressed the expression of PPARG in ER-positive AN3 CA and promoted the expression of PPARG in ER-negative HEC-1-A. D–I Kaempferol suppressed the expression of PPARGC1A and ESRRA in both AN3 CA (D–F) and HEC-1-A cells (G–I), without modulating ESR1. Western blotting (D–E and G–H) and the IHC scores (F and I) confirmed the differential expression of PPARGC1A and ESRRA. Results are presented as means and SDs. Compared with the negative control, *, #P < 0.05, **, ##P < 0.01, ***, ###P < 0.001

Article Snippet: The whole cell lysates and tumor homogenates (50 μg) were resolved on an 8 ~ 12% SDS–polyacrylamide gel, transferred to a polyvinylidene difluoride membrane (NEN Life Sciences, Boston, MA), probed sequentially with antibodies against ESR1 (ab108398, 67 kDa), ESRRA (ab137489, 55 kDa), PPARGC1A (ab188102, 91 kDa) (Abcam, Cambridge, MA, U. S.), CASP3/p17/p19 (19677–1, 35 kDa), CASP9/p35/p10 (66169–1, 46 kDa), PPARG (16643–1, 55 kDa), HSD17B1 (25334–1, 35 kDa) (Proteintech, Wuhan, China) at 4 °C overnight, rinsed, and incubated with the goat anti-rabbit secondary antibody (Abcam, Cambridge, MA).

Techniques: Expressing, Binding Assay, Activity Assay, Western Blot, Quantitative Proteomics, Negative Control

Journal: Frontiers in Nutrition

Article Title: Zhaqu compound improves glucose and lipid metabolism in T2DM with MASLD by modulating gut microbiota and PPARγ

doi: 10.3389/fnut.2026.1775686

Figure Lengend Snippet: ZQC regulates lipid metabolism via the PPARγ pathway in HG/PA-induced HepG2 cells. (A,B) Intracellular TG and T-CHO levels. (C,D) Relative expression of proteins (e.g., SREBP-1c, ACC1). (E–H) WB quantification of representative lipogenic and PPARγ pathway proteins (FASN, PPARγ, CD36, FABP4) in control, HG/PA, HG/PA + 5% ZQC-S, and HG/PA + 7.5% ZQC-S groups. (I) Representative Oil Red O staining images of lipid droplets. (J) Quantification of lipid droplet content (ORO%) in each group. (K,L) Intracellular TG and T-CHO levels. (M) WB analysis of key proteins in control, HG/PA, HG/PA + 7.5% ZQC-S, and HG/PA + 7.5% ZQC-S + GW1929 groups. (N–S) Relative expression of selected lipid metabolism and PPARγ pathway proteins across the four groups. ( # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CON; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. HG/PA; & p < 0.05, && p < 0.01, &&& p < 0.001 vs. HG/PA + 5% ZQC-S + g1929).

Article Snippet: The reagents and antibodies used were the Total Protein (TP) Assay Kit (1,000 tests, P0006, Biyuntian Biotechnology, China), Glucose Assay Kit (96 T, A154-1-1, Nanjing Jiancheng Bioengineering Institute, China), Total Cholesterol (T-CHO) Assay Kit (96 T, A111-1-1, Nanjing Jiancheng Bioengineering Institute, China), Triglyceride (TG) Assay Kit (96 T, A110-1-1, Nanjing Jiancheng Bioengineering Institute, China), Sequencing Reagent Kit (NovaSeq 6,000 SP Reagent Kit V1.5, Illumina, USA), RNA Mini Kit (Qiagen, Germany), DMEM High Glucose Medium (PM150210, Procell, China), Trypsin (S310JV, Shanghai Yuanpei, China), Fetal Bovine Serum (C04001-500, Vivacell, China), Double Antibody (S110JV, Shanghai Yuanpei, China), PPARγ agonist (HY-146480, MCE, USA), Palmitic acid (H8780, Solarbio, China), Oil Red O staining kit (G1262, Solarbio, China), 2-NBDG fluorescent probe (HY-116215, MCE, USA), BCA protein concentration assay kit (BL521C, Biosharp, China), SDS-PAGE Protein Loading Buffer (5×) (BL502A, Biosharp, China), ECL Chemiluminescent Substrate (BL520B, Biosharp, China), Western Blot & IP Cell Lysis Buffer (P0013, Beyotime, China), PBS Buffer ( PB180327 , Procell, China), and the primary antibodies β -Microtubulin (AC026, Abclonal, China), PEPCK (ET7107-29, Huabio, China), G6Pase (A21168, Abclonal, China), GLUT2 (A12307, Abclonal, China), SREBP-1C (ER1917-19, Huabio, China), ACC1 (ET1609-77, Huabio, China), FASN (R1706-8, Huabio, China), PPARγ (A19676, Abclonal, China), CD36 (ET1701-24, Huabio, China), and FABP4 (ET1703-98, Huabio, China).

Techniques: Expressing, Control, Staining

Journal: Free radical biology & medicine

Article Title: Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling.

doi: 10.1016/j.freeradbiomed.2020.06.005

Figure Lengend Snippet: Fig. 1. PPARγ2 and Tnpo1 bind via disulfide bonds. (A–C) Six-week-old male C57BL/6 mice were intravenously injected via the tail vein with 4 × 109 plaque-forming units of mock adenovirus or PPARγ2 adenovirus (adPPARγ2). Livers were dissected 2 weeks later for analysis (n = 3/group). (A) Hepatic TG levels. **P < 0.01 vs. mice infected with mock adenovirus. (B) Immunoprecipitation of PPARγ2/Tnpo1, PPARγ2/importin-α, and PPARγ2/importin-β complexes from liver samples. (C) Immunoprecipitation of PPARγ2/Tnpo1 complex from liver samples with or without 10 mM DTT treatment. (D–F) Immunoprecipitation of PPARγ2:ΔCys (Ser→Cys)/Tnpo1 (D), PPARγ2:WT (Cys→Ser)/ Tnpo1 (E), and PPARγ2/Tnpo1:WT (Cys→Ser) complexes (F). (G) PPARγ2/Tnpo1 complex (yellow), PPARγ2 (green), and Tnpo1 (red) in mouse primary cultured hepatocytes transfected with PPARγ2 or PPARγ2:ΔCys expression vector for 48 h, then treated with 250 μM H2O2 or left untreated for 6 h. Cell lysates were immunoprecipitated with anti-PPARγ antibody, treated with 10 mM DTT or left untreated, and then subjected to non-reducing sodium dodecyl sulphate–polya- crylamide gel electrophoresis followed by western blotting. All data are expressed as mean ± SEM. P value was calculated using unpaired Student's t-test in A. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mutant mouse Tnpo1 and PPARγ2 expression vectors were prepared using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) from MycDDK-tagged mouse Tnpo1 and PPARγ2 expression vectors (Origene).

Techniques: Injection, Infection, Immunoprecipitation, Cell Culture, Transfection, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot

Journal: Free radical biology & medicine

Article Title: Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling.

doi: 10.1016/j.freeradbiomed.2020.06.005

Figure Lengend Snippet: Fig. 3. Cytosolic H2O2/Tnpo1-dependent nuclear translocation of PPARγ2 enhances PPARγ2 target gene expression and TG accumulation in hepatocytes. (A–C) WT and Sod1-deficient hepatocytes were infected with mock adenovirus or adPPARγ2 at a MOI of 50 for 48 h, followed by culturing with or without 5 μM DMNQ for 6 h. (D–F) WT hepatocytes were infected with mock adenovirus or adPPARγ2 at an MOI of 50, transfected with Tnpo1 or control siRNA for 24 h, and cultured with or without 5 μM DMNQ for 6 h. (A, D) (Left) Oil Red O staining. (Right) Quantification of Oil Red O staining. (B, C, E, F) Quantification of Pparg2, Fsp27, Fabp1, and Fabp4 mRNA levels (B, E) and the amount of DNA-bound PPARγ (C, F) in WT and Sod1-deficient hepatocytes (n = 5/group). *P < 0.05, **P < 0.01 vs. WT hepatocytes infected with mock adenovirus without DMNQ treatment (A–C) and vs. hepatocytes infected with mock adenovirus, transfected with control siRNA, and without DMNQ treatment (D–F). All data are expressed as mean ± SEM. P value was calculated by one-way ANOVA in A, B, C, D, E, and F. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mutant mouse Tnpo1 and PPARγ2 expression vectors were prepared using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) from MycDDK-tagged mouse Tnpo1 and PPARγ2 expression vectors (Origene).

Techniques: Translocation Assay, Targeted Gene Expression, Infection, Transfection, Control, Cell Culture, Staining

Journal: Free radical biology & medicine

Article Title: Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling.

doi: 10.1016/j.freeradbiomed.2020.06.005

Figure Lengend Snippet: Fig. 4. Cytosolic H2O2/Tnpo1-dependent nuclear translocation of PPARγ2 promotes hepatic TG accumulation in mice infected with adPPARγ2. (A–F) Six-week-old male C57BL/6 WT and Sod1-deficient mice were intravenously injected via the tail vein with 4 × 109 plaque-forming units of mock adenovirus and PPARγ2 and SOD1 adenoviruses (adPPARγ2 and adSOD1, respectively) in various combinations. Livers were dissected from mice 2 weeks later for analysis. (G–J) Six-week-old male C57BL/6 WT and Sod1-deficient mice were intravenously injected via the tail vein with 4 × 109 plaque-forming units of mock adenovirus or adPPARγ2, followed by injection of control or Tnpo1 siRNA (1 mg/kg body weight) after 2 days. Livers were dissected from mice 12 days later for analysis. (A, G) Representative H&E-stained liver sections. (F) Immunoprecipitation of PPARγ2/Tnpo1 complex from liver tissue. (B–E, H–J) Quantification of hepatic TG (B, H), cytosolic H2O2 (C), and Pparg2, Fsp27, Fabp1, and Fabp4 mRNA (D, I) levels and the amount of DNA-bound PPARγ (E, J) (n = 5/group). **P < 0.01 vs. WT mice infected with mock adenovirus (B–E); and *P < 0.05, **P < 0.01 vs. WT mice infected with mock adenovirus and treated with control siRNA (H–J). All data are expressed as mean ± SEM. P value was calculated by one-way ANOVA in B, C, D, E, H, I, and J.

Article Snippet: Mutant mouse Tnpo1 and PPARγ2 expression vectors were prepared using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) from MycDDK-tagged mouse Tnpo1 and PPARγ2 expression vectors (Origene).

Techniques: Translocation Assay, Infection, Injection, Control, Staining, Immunoprecipitation

Journal: Free radical biology & medicine

Article Title: Redox-dependent PPARγ/Tnpo1 complex formation enhances PPARγ nuclear localization and signaling.

doi: 10.1016/j.freeradbiomed.2020.06.005

Figure Lengend Snippet: Fig. 5. Cytosolic H2O2/Tnpo1-dependent nuclear translocation of PPARγ2 promotes hepatic TG accumulation in mice fed an HFD. (A–F) Six-week-old male C57BL/6 mice were fed a control diet or HFD for 4 weeks, then intravenously injected via the tail vein with control, Tnpo1, Sod1, or Pparg2 siRNA (1 mg/kg body weight) in various combinations. Livers were dissected from the mice 10 days later for analysis (n = 5/group). (A) Representative H&E-stained liver sections. (B–D) Quantification of TG (B), cytosolic H2O2 (C), and Pparg2, Fsp27, Fabp1, and Fabp4 mRNA levels (D) in the liver (n = 5/group). **P < 0.01 vs. mice fed a control diet and treated with control siRNA. (E) Immunoprecipitation of PPARγ2/Tnpo1 complex in the liver. (F) Quantification of the amount of DNA- bound PPARγ in the liver (n = 5/group). **P < 0.01 vs. mice fed a control diet and treated with control siRNA. #P < 0.05, ##P < 0.01 vs. mice fed an HFD diet and treated with control siRNA. All data are expressed as mean ± SEM. P value was calculated by one-way ANOVA in B, C, D, E, and F.

Article Snippet: Mutant mouse Tnpo1 and PPARγ2 expression vectors were prepared using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) from MycDDK-tagged mouse Tnpo1 and PPARγ2 expression vectors (Origene).

Techniques: Translocation Assay, Control, Injection, Staining, Immunoprecipitation

Journal: Journal of the Endocrine Society

Article Title: Forkhead Box Transcription Factors of the FOXA Class Are Required for Basal Transcription of Angiotensin-Converting Enzyme 2

doi: 10.1210/js.2016-1071

Figure Lengend Snippet: Forkhead box transcription factors bind to the ACE2 proximal promoter region. (a) An EMSA was conducted with the R4 DNA probe. Nuclear extracts were from untransfected 832/13 cells or 832/13 cells transfected with COUP-TFII or PPAR γ expression plasmids. Antibodies against COUP-TFII and PPAR γ were included in the binding reactions as indicated. (b) The R6 region has similarity to FOXO1 and FOXA1 motifs as indicated by the BKL TRANSFAC program, whereas the mutation destroys the similarity. (c) An EMSA was done with the R6 probe and nuclear extracts from 832/13 cells that were untransfected or transfected with a FOXO1 expression plasmid (left panel). The right panel shows the effect on the band pattern when an antibody against FOXO1 is included. (d) An EMSA was done with the R6 probe and nuclear extracts from 832/13 cells that were untransfected or transfected with FOXA1, FOXA2, or FOXA3 expression plasmids (left panel). The right panel shows the effects on the band pattern when antibodies against the FOXA transcription factors are included in the binding reactions. (e) An EMSA was conducted with a nuclear extract from 832/13 cells. The probes were the human and mouse R6 regions as well as regions in the distal promoter region with a putative FOXA binding site. An antibody recognizing FOXA1 and FOXA2 was included in the binding reactions as indicated.

Article Snippet: A plasmid containing the open reading frame for human PPAR γ was purchased from Origene (SC 124177).

Techniques: Transfection, Expressing, Binding Assay, Mutagenesis, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Identification of PTGR2 inhibitors as a new therapeutic strategy for diabetes and obesity

doi: 10.1038/s44321-025-00216-4

Figure Lengend Snippet: ( A ) Metabolism of 15-keto-PGE2. ( B ) Activation of murine PPARγ (mPPARγ) measured by Gal-PPARγ/UAS-LUC reporter assay in HEK293T cells ( n = 3 per group, 3 biological replicates with 1 technical replicate each). Cells were transfected with Gal4-PPARγ, UAS-LUC, and TK-Rluc (Renilla luciferase), and treated with pioglitazone (** P = 0.0021, **** P < 0.0001, *** P = 0.0002) or 15-keto-PGE2 (** P = 0.0015, * P = 0.0137, ** P = 0.0014). RT-qPCR of ( C ) Glut4 (**** P < 0.0001, ** P = 0.0005, **** P < 0.0001) and other mPPARγ-downstream genes including ( D ) Irs2 (**** P < 0.0001, * P = 0.0475, **** P < 0.0001), ( E ) Sorbs1 (**** P < 0.0001, ** P = 0.0023), ( F ) Cd36 (*** P = 0.0002, *** P = 0.0006), ( G ) Acs (**** P < 0.0001, ** P = 0.0029), ( H ) Cepba (** P = 0.0016, * P = 0.0274, ** P = 0.0025), and ( I ) Adipoq (** P = 0.0023, **** P < 0.0001, **** P < 0.0001) in differentiated 3T3-L1 adipocytes treated with 15-keto-PGE2 ( n = 3 per group, 3 biological replicates with 2 technical replicate each). ( J ) Effect of 15-keto-PGE2 on insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes (**** P < 0.0001, *** P = 0.0002, **** P < 0.0001; n = 4 per group, 4 biological replicates with 1 technical replicate each). ( K ) HEK293T cells transfected by mPPARγ and treated with 15-keto-PGE2. Covalent binding of 15-keto-PGE2 to mPPARγ detected by liquid-chromatography tandem mass spectrometry (LC-MS/MS). ( L ) Reciprocal co-immunoprecipitation of mPPARγ and cysteine-15-keto-PGE2. Myc-DDK-mPPARγ and Myc-DDK-mPPARγ C313A were expressed in HEK293T cells, and immunoprecipitation (IP) conducted using either anti-DDK (anti-Flag) or anti-15-keto-PGE2-cysteine-BSA antibody, followed by immunoblotting with anti-15-keto-PGE2-cysteine-BSA and anti-DDK antibody. ( M ) PPRE reporter activity after addition of 15-keto-PGE2 to HEK293T cells transfected with wild-type and C313A mutant mPPARγ (** P = 0.0037, ** P = 0.0077, **** P < 0.0001, **** P < 0.0001; n = 3 per group, 3 biological replicates with 1 technical replicate each). ( N ) Native mass spectrometry spectrum showed the binding of 15-keto-PGE2 to wild-type and mPPARγ mutants (C313A and H351A). The spectrum of unbound free-form proteins was shown in the left panel. The spectrum of bound form after the addition of 15-keto-PGE2 was shown in the right panel ( n = 4 per group, 4 independent experiments with 1 technical replicate each) and ( O ) histogram (**** P < 0.0001). ( P ) 15-keto-PGE2 enhanced insulin-stimulated glucose uptake in PPARγ-null 3T3-L1 clones (#1296; n = 4 per group, 4 biological replicates with 1 technical replicate each) rescued with wild-type mPPARγ (**** P < 0.0001, ** P = 0.0036) but not in those rescued with mutant mPPARγ (C313A) (**** P < 0.0001). ( Q ) Diagram showing the motifs of mPPARγ and 15-keto-PGE2 binding site. Data information: Data are presented as mean and standard error (S.E.M.). Statistical significance was calculated by one-way analyses of variance (ANOVA) with Tukey’s post hoc test in ( B – J , P ) and two-sample independent t -test in ( M , O ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns means no statistical difference. .

Article Snippet: Murine PPARγ plasmid , OriGene, USA , Cat.# MC201042.

Techniques: Activation Assay, Reporter Assay, Transfection, Luciferase, Quantitative RT-PCR, Binding Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Western Blot, Activity Assay, Mutagenesis, Clone Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Abscisic acid regulates dormancy of prostate cancer disseminated tumor cells in the bone marrow

doi: 10.1016/j.neo.2020.11.009

Figure Lengend Snippet: ABA induces cellular dormancy through PPARγ receptor signaling in PCa cells. (A) Basal levels of mRNA expression of the receptor of ABA, PPARγ in PCa cell lines as quantified by real-time PCR. (B) Basal levels of protein expression of the receptor of ABA, PPARγ in PCa cell lines as quantified by Western blot. (C) mRNA expression of PPARγ in G 0 , G 1 , and S/G 2 /M cell cycle phase of PC3 VC cells. (D) Verification of PPARγ mRNA expression in PPARγ silencing PC3 VC as quantified by real-time PCR. (E) Proliferation assays in PC3 VC -Control or PC3 VC -shPPARγ cells were performed in 1% FBS culture condition with ABA treatment. (F)% of G 0, G 1, or S/G 2 /M cell cycle phase in PC3 VC -Control or PC3 VC -shPPARγ cells following vehicle or ABA (50 µM) treatment in 1% FBS culture conditions at 72 h as quantified by FACS analyses. Data in C–F are representative of mean with SD (Student's t test). (G) Activation of down-stream effector, p70S6K of mTOR signaling in PC3 VC -Control or PC3 VC -shPPARγ cells following vehicle or ABA (50 µM) treatment as quantified by Western blot.

Article Snippet: Lentiviral particles with PPARγ human shRNA or negative control (cat no. TL320459V , Origene, Rockville, MD) were infected into PC3 VC cells in the presence of 5 μg/mL polybrene for 48 h. Infected cells were selected for 7 d in media containing 1 μg/mL Puromycin and analyzed by real-time PCR or ELISA.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control, Activation Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Abscisic acid regulates dormancy of prostate cancer disseminated tumor cells in the bone marrow

doi: 10.1016/j.neo.2020.11.009

Figure Lengend Snippet: ABA and PPARγ signaling pathway induces PCa cellular dormancy in the bone marrow microenvironment. (A) ABA production was identified in culture media of MC3TC-E1 cells at 72 h as quantified by ELISA (cat. LS- F4483-1, Life Span Biosciences). ABA production was normalized to total protein. (B) % of G 0, G 1, or S/G 2 /M cell cycle phase when PC3 VC -Control or PC3 VC -shPPARγ cells are co-cultured with murine osteoblasts (MC3T3-E1 cells) with or without ABA (50 µM) treatment. Live cells (cat. NBP2-31156, DAPI, NOVUS) were negatively gated for anti-mouse H-2kd (cat no. 116622, PE/Cy7, BioLegend), which were then positively gated for (human) HLA-A,B,C (cat no. 311426, APC/Cy7, BioLegend). After these gates were applied, the cells were plotted on the Venus-Cherry spectrum. Cell cycle phase was determined using FACS analyses. (C) Diagram of the experimental procedures for in vivo animal model. PC3 VC -Control or PC3 VC -shPPARγ cells (2 × 10 5 cells) were suspended in 30μl of PBS and injected into 5- to 7-wk-old male CB.17. SCID mice by i.t. injection. After PCa cell injection, vehicle or ABA (20 mg/kg) treatment for 8 times (twice daily) was followed by i.p. injection. At 4 d, mice were sacrificed, and the tibiae which PCa cells were injected were collected for analyzing cell cycle phase of PC3 VC cells by FACS analyses. Antibody staining were applied as same as in coculture study in A. (D) Quantification of % of G 0, G 1, or S/G 2 /M cell cycle phase from 5C ( n = 4/group). Data in A, B and D are representative of mean with SD (Student's t test).

Article Snippet: Lentiviral particles with PPARγ human shRNA or negative control (cat no. TL320459V , Origene, Rockville, MD) were infected into PC3 VC cells in the presence of 5 μg/mL polybrene for 48 h. Infected cells were selected for 7 d in media containing 1 μg/mL Puromycin and analyzed by real-time PCR or ELISA.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Cell Culture, In Vivo, Animal Model, Injection, Staining