Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Relieving colitis by miR-223 through the promotion of macrophage M2 polarization via the modulation of PPAR-γ/FOXO1 signaling. (A) Representative images of western blots indicating the protein expression levels of PPAR-γ and FOXO1. The data represent the findings from three independent experiments. (B, C) Changes in the expression of PPAR-γ and FOXO1. (D, E) Changes in the mRNA levels of PPAR-γ and FOXO1. All the data are expressed as the means ± SDs (n=6 each group). *p<0.05, **p<0.01, ***p<0.001. (F, G) Correlations of colonic miR-223 expression with PPAR-γ and FOXO1 expression in the colon. Pearson analyses were used to correlate miR-223 with colonic PPAR-γ and FOXO1 expression. PPAR-γ, peroxisome proliferator-activated receptor gamma; FOXO1, Forkhead box transcription factor O1.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Immunofluorescence analysis of PPAR-γ and M2 macrophage marker CD206 in colonic tissue. Representative micrographs depict protein expression levels of PPAR-γ (red) and its co-localization (yellow) with CD206 (green) in colon sections. Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques: Immunofluorescence, Marker, Expressing

Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Schematic representation of the potential mechanisms underlying the targeted therapy of miR-223 supplement for DSS-induced colitis. miR-223 ameliorates DSS-induced colitis through promoting macrophage M2 polarization via modulation of PPAR-γ and FOXO1 signaling. DSS, dextran sodium sulfate.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

doi: 10.1371/journal.pone.0045514

Figure Lengend Snippet: Oligonucleotides and plasmids.

Article Snippet: pSV Sport PPARγ , PPARγ expression plasmid , Addgene 8886, .

Techniques: Luciferase, Plasmid Preparation, Expressing, Control, Dominant Negative Mutation

Journal: PLoS ONE

Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

doi: 10.1371/journal.pone.0045514

Figure Lengend Snippet: Flotillin promoter constructs F1-1330 (A, C) or F2-2130 (B, D) were cotransfected into Hela cells together with expression plasmids for RAR, RXR, PPARγ or with empty PSV control plasmid. One day post-transfection, the cells were stimulated with trans-RA (1 µM) for 24 h in serum-free medium. Relative luciferase activity of the unstimulated control sample was set as 1. F1-1330 (E) and F2-2130 (F) transfected Hela cells were stimulated with troglitazone for 24 h in serum-free medium. Values are mean ± standard deviation of at least 3 experiments measured in duplicates. ***p<0.001; **p<0.01; *p<0.05 vs. respective control.

Article Snippet: pSV Sport PPARγ , PPARγ expression plasmid , Addgene 8886, .

Techniques: Construct, Expressing, Control, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Transcriptional Regulation of Flotillins by the Extracellularly Regulated Kinases and Retinoid X Receptor Complexes

doi: 10.1371/journal.pone.0045514

Figure Lengend Snippet: Hela cells were transiently transfected with expression constructs for RAR, RXR, or a combination of both. Empty PSV vector served as a control. One day post-transfection, the cells were stimulated with trans-RA (1 µM) in serum-free medium for 24 h. Cell lysates were analyzed for flotillin-1 (A), flotillin-2 (B), RAR and RXR (C) by Western blotting. D and E show a densitometric quantification of flotillin expression. F: Cells were transfected with RAR or PPARγ expression construct or empty PSV. RNA was isolated, transcribed into cDNA and flotillin mRNA was measured by qPCR. Values are mean ± standard deviation of at least 3 experiments. ###, p<0.001; #, p<0.05; vs control *, p<0.05 vs. unstimulated sample.

Article Snippet: pSV Sport PPARγ , PPARγ expression plasmid , Addgene 8886, .

Techniques: Transfection, Expressing, Construct, Plasmid Preparation, Control, Western Blot, Isolation, Standard Deviation

Journal: Journal of lipid research

Article Title: Disruption of nucleotide biosynthesis reprograms mitochondrial metabolism to inhibit adipogenesis.

doi: 10.1016/j.jlr.2024.100641

Figure Lengend Snippet: Fig. 4. PPARγ overexpression rescues mitochondrial function induced by the loss of de novo nucleotide biosynthesis. A: OXPHOS protein levels were measured in primary SVF cells that were differentiated and treated with PPARγ inhibitor SR 16832 (2 μM or 10 μM) for 6 days (B) 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM 5FU or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. C: 3T3-L1 cells stably expressing pBABE control vector or PPARγ2 were differentiated and treated with 10 μM MIZ or DMSO (control) for 6 days. Protein expression was analyzed by Western blot as indicated. D: Live cell imaging with MTG and TMRE staining was used to assess changes in mito- chondrial membrane potential relative to mitochondrial mass in the presence or absence of 25 μM MIZ. E: ImageJ was used to measure the ratio of MTG and TMRE. All experiments were repeated two or three times with two or three biological replicates. Statistical significance was determined using one-way ANOVA multiple comparisons test. Error bars indicate mean ± SD, ****P < 0.0001.

Article Snippet: PPARγ2 plasmid (Addgene #8859) or pBabe control was transfected into PLAT-E cells.

Techniques: Over Expression, Stable Transfection, Expressing, Control, Plasmid Preparation, Western Blot, Live Cell Imaging, Staining, Membrane

Journal: Oncogene

Article Title: A Novel Interaction of PAK4 with PPARγ to Regulate Nox1 and Radiation-Induced Epithelial-to-Mesenchymal Transition in Glioma

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: (A) Identification of potential PAK4 associating TFs using TF-TF Interaction Array. PAK4 was immunoprecipitated from nuclear extracts of 4910 cells with anti-PAK4 antibody and used as a bait. Isotype-specific IgG precipitates were used as negative control. PAK4 association with various TFs is identified as horizontal duplicate spots on the x-ray film. “+” indicates the positive control signals. (B) 4910 cells were treated with EV and PAK4-FL for 48 hours and IP experiments was performed with nuclear lysates (500 µg) from 4910 cells with specific antibodies against PAK4 and non-specific IgG followed by immunoblotting with PPARγ. Inputs indicate 10% of each pre-IP samples. (C) PPARγ IP using anti-FLAG and anti-IgG antibody from nuclear lysates of 4910 cells at 48 hours post-transfection with EV or FLAG-PPARγ constructs followed by immunoblotting for PAK4. (D) IP experiments using 4910 lysates with either a PAK4 specific antibody or non-specific IgG followed by immunoprobing for PPARγ. Reciprocal IPs were performed with anti-PPARγ antibody and subsequent immunoblotting with PAK4 to confirm PAK4/PPARγ association in the nucleus. (E) Identification of minimal PPARγ-interaction domain of PAK4 using bacterially expressed GST, and GST-PPARγ purified using MagneGST Pull-Down System following manufacturer’s protocol. Biotin-labeled PAK4 truncated mutants (1–290aa, 291–591aa 1–591aa) were incubated with GST-PPARγ, separated on 10% SDS-PAGE and detected as described in Materials and Methods (Top panel). Inputs (10% samples) were analyzed by SDS-PAGE (Bottom panel). (F) Schematic representation of mapping PPARγ interacting domain on PAK4 using different truncation mutants. CRIB: Cdc42-and Rac-interactive binding motif; GID: GEF-H1 Interaction Domain; KD: Kinase Domain. (G) EMSA was performed with 4910 Nuclear extracts (5µg) to detect PPARγ activity. For the supershift analyses, specific antibodies against PAK4 and PPARγ were incubated with control sample before adding the biotin-labeled probe.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD), kinase-dead PAK4 plasmid (PAK4-K350M), PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa) (PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549) , FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895) and respective empty vector controls were used in the study.

Techniques: Immunoprecipitation, Negative Control, Positive Control, Western Blot, Transfection, Construct, Purification, Labeling, Incubation, SDS Page, Binding Assay, Activity Assay

Journal: Oncogene

Article Title: A Novel Interaction of PAK4 with PPARγ to Regulate Nox1 and Radiation-Induced Epithelial-to-Mesenchymal Transition in Glioma

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: (A) IP with antibodies against non-specific IgG and PPARγ using nuclear lysates prepared from control and IR (8Gy)-treated 4910 and 5310 cells followed by immunoprobing with PAK4 antibody (Top panels). Reciprocal IPs were performed with anti-IgG and anti-PAK4, and subsequently immunoprobed with PPARγ to confirm changes in radiation-induced PAK4/PPARγ interaction in these cells (Bottom panels). Representative blots from three independent experiments are shown. (B) Sub-cellular localization analyses of PAK4 (green) and PPARγ (red) by confocal microscopy in control and IR-treated cells. Scale bars: 10µm. (C) Protein-DNA array (version II) interaction analysis performed with immunoprecipitated PAK4 from 4910 nuclear extracts as described in Materials and Methods. PAK4 binding with TF-consensus sequences was detected by duplicate spots on the membrane. “+” indicates positive control signal.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD), kinase-dead PAK4 plasmid (PAK4-K350M), PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa) (PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549) , FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895) and respective empty vector controls were used in the study.

Techniques: Confocal Microscopy, DNA Array, Immunoprecipitation, Binding Assay, Positive Control

Journal: Oncogene

Article Title: A Novel Interaction of PAK4 with PPARγ to Regulate Nox1 and Radiation-Induced Epithelial-to-Mesenchymal Transition in Glioma

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: (A) Real-time PCR showing Nox1 transcriptional levels in control- and IR-treated cells. The fold change values are represented as mean ± SD (n=5) obtained from at least three independent experiments (*p≤0.01). (B) Immunoblotting shows Nox1 expression with GAPDH served as an internal control. (C) Whole cell lysates were subjected immunoblotting and representative blots from three independent experiments were shown. (D) Schematic representation of putative PPARγ binding sites on Nox1 promoter. Seven putative PPRE sites were identified located in the promoter (4 sites), exon-1 (1 site) and intron-1 (2 sites) of human Nox1 based on analysis of a 2.9-kb 5’-flanking region of Nox1 (GenBank: ABC40742.1). ChIP primers specific for R-1, R-2, R-3 and R-4 regions (blue arrows) were used to determine PPARγ recruitment on Nox1 promoter. (E) ChIP analysis of PPARγ occupancy around PPREs on the Nox1 promoter using DNA from 4910 and 5310 cells and IP with anti-IgG and anti-PPARγ antibodies with and without IR treatment. 5% of pre-ChIP DNA samples served as input controls for each sample. ChIP-DNA from control and IR-treated cells were analyzed by quantitative PCR using ChIP-specific primers covering Nox1 promoter regions (R-1 to R-4). ChIP amplification is shown as percent input from three different experiments (n=5) (*p≤0.05, **p≤0.01). (F) ChIP assay was performed with R-2 primers and antibodies against non-specific IgG and PPARγ using ChIP-DNA as described above in both 4910 and 5310 cells. Subsequently, re-ChIP assay was performed using primary ChIP amplicons with anti-IgG and anti-PAK4 antibodies; results from three experimental replicates are shown.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD), kinase-dead PAK4 plasmid (PAK4-K350M), PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa) (PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549) , FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895) and respective empty vector controls were used in the study.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Binding Assay, Amplification

Journal: Oncogene

Article Title: A Novel Interaction of PAK4 with PPARγ to Regulate Nox1 and Radiation-Induced Epithelial-to-Mesenchymal Transition in Glioma

doi: 10.1038/onc.2016.261

Figure Lengend Snippet: (A) Paraffin-embedded brain tumor sections were stained and tumor volumes were measured as described in Materials and Methods. Relative tumor size is shown as mean ± SD obtained from different groups as indicated (n=6) (*p≤0.05, **p≤0.01). (B) Immunohistochemical analysis of brain tumors from nude mice that were intracranially implanted with SV or PAK4sh cells and subjected to IR treatments as described in Materials and Methods; representative micrographs are shown. Inset: staining with Non-specific IgG. (C) Confocal microscopy was performed in tumor sections to determine N-cadherin (red) and E-cadherin expression (green) levels. (D) Schematic diagram represents the radiation-induced PAK4 nuclear translocation, binding with PPARγ and co-recruitment of PAK4/PPARγ complex on to Nox1 promoter which further results in Nox1 transactivation, ROS generation and EMT induction in glioma cells.

Article Snippet: The human PAK4.shRNA plasmid (PAK4sh) comprising a pool of three different targeting shRNAs (sc-39060-SH) and specific scrambled shRNA vector (SV) (Santa Cruz Biotechnology, Santa Cruz, CA), full-length GFP-tagged PAK4 overexpression plasmid (PAK4-FL) (Origene, Rockville, MD), kinase-dead PAK4 plasmid (PAK4-K350M), PAK4 NLS mutant plasmid (NLS1, Lysine mutated to Alanine within 4–8 aa) (PAK4-NLS-Mut), GST-tagged PPARγ (GST-PPARγ; Addgene plasmid 16549) , FLAG-tagged PPARγ (FLAG-PPARγ; Addgene plasmid 8895) and respective empty vector controls were used in the study.

Techniques: Staining, Immunohistochemical staining, Confocal Microscopy, Expressing, Translocation Assay, Binding Assay

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