Journal: Animals : an Open Access Journal from MDPI

Article Title: Chicken Protein S Gene Regulates Adipogenesis and Affects Abdominal Fat Deposition

doi: 10.3390/ani12162046

Figure Lengend Snippet: PROS1 inhibits chicken preadipocyte differentiation. ( A ) The oil red O staining and statistics of ICP-1 after treating with pcDNA.1- PROS1 plasmid and si- PROS1 . ( B , C ) The mRNA levels of preadipocyte differentiation-related genes in ICP-1 treated with PROS1 overexpression and knockdown were detected by qRT-PCR. ( D , E ) The protein levels of CEBPα and PPARγ in ICP-1 treated with PROS1 overexpression and knockdown were detected by Western blot. ( F ) The glucose consumption measure during ICP-1 differentiation. ( G ) The glycerol production measure during ICP-1 differentiation. The results are all presented as means ± SEM, * p < 0.05. Original western blot figures in .

Article Snippet: Then, the membrane was incubated with anti-PPARγ (1:1000; bs-0530R, BIOSS), anti-CEBPα (1:1000; LS-B4685, LABIO), or anti-GAPDH antibodies (1:2000; bsm-33033M, BIOSS) at 4 °C overnight.

Techniques: Staining, Plasmid Preparation, Over Expression, Quantitative RT-PCR, Western Blot

Journal: Scientific Reports

Article Title: RXR Expression in Marine Gastropods with Different Sensitivity to Imposex Development

doi: 10.1038/s41598-020-66402-1

Figure Lengend Snippet: RXR and PPARγ expression in males of Buccinanops globulosus and Trophon geversianus . ( a ) Immunohistochemical staining for RXR ( top ) and PPARγ ( bottom ) in sections of paraffin-embedded penis of Buccinanops globulosus and Trophon geversianus males collected in CA.VD: vas deferens. Bar: 50 µm. ( b ) Control assays from ( a ) in which no primary antibody was added. VD: vas deferens. Bar: 50 µm. ( c ) Quantification of positive cells/HPF from experiments depicted in ( a ). ***p < 0.001. B.g .: Buccinanops globulosus , T.g .: Trophon geversianus . ( d ) Representative western blots showing RXR ( top left ) and PPARγ ( bottom left ) expression in total extracts from penis of Buccinanops globulosus ( B.g .) and Trophon geversianus ( T.g .) males collected in CA. β-Actin was used as a loading control. The band intensity ratios of RXR and PPARγ expression relative to β–Actin were plotted ( right , ***p < 0.001). Mouse lung and kidney tissues were used as positive controls. Full-length blots are presented in Supplementary Fig. .

Article Snippet: Membranes were probed with RXR (sc-774), PPARγ (sc-7273) or β-Actin (sc-47778) antibodies (Santa Cruz Biotechnology) overnight at 4 °C, and then incubated with horse anti-mouse or goat anti-rabbit peroxidase-conjugated secondary antibody (Vector Laboratories).

Techniques: Expressing, Immunohistochemical staining, Staining, Control, Western Blot

Journal: Scientific Reports

Article Title: RXR Expression in Marine Gastropods with Different Sensitivity to Imposex Development

doi: 10.1038/s41598-020-66402-1

Figure Lengend Snippet: RXR and PPARγ expression in imposex-affected females. ( a ) Immunohistochemical staining for RXR in sections of paraffin-embedded penis of Buccinanops globulosus males and imposex-affected females collected in CA and LPBH.VD: vas deferens. Bar: 50 µm. ( b ) Quantification of RXR positive cells/HPF from experiments depicted in ( a ). ***p < 0.001. ( c ) Representative western blots showing RXR and PPARγ ( left ) expression in total extracts from penis of Buccinanops globulosus males and imposex-affected females collected in CA and LPBH. β-Actin was used as a loading control. The band intensity ratios of RXR and PPARγ expression relative to β–Actin were plotted ( middle and right respectively). Full-length blots are presented in Supplementary Fig. .

Article Snippet: Membranes were probed with RXR (sc-774), PPARγ (sc-7273) or β-Actin (sc-47778) antibodies (Santa Cruz Biotechnology) overnight at 4 °C, and then incubated with horse anti-mouse or goat anti-rabbit peroxidase-conjugated secondary antibody (Vector Laboratories).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Control

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: 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 biochemistry

Article Title: PPARα activation partially drives NAFLD development in liver-specific Hnf4a-null mice.

doi: 10.1093/jb/mvad005

Figure Lengend Snippet: Fig. 5. Promoter analysis of the Fatp1 gene by PPARs and the binding of PPARs to PPRE of the Fatp1. (A) Schematic diagram of the promoter of the Fatp1 gene. PPAR response element (PPRE) locates at −542/−444 from the transcrition start site. Promoter constructs of promoterless, the Fatp1 gene with and without PPRE (−444/+52; (-PPRE) and −542/+52; (+PPRE)) were transfected into HEK293T cells. At that time, PPARα (B), PPARβ/δ (C), and PPARγ (D) expression vector was co-transfected with PGC1α expression vector in the absence and presence of the ligand of PPARα (10 μM Wy-14,643), PPARβ/δ (100 nM GW501516), and PPARγ (5 μM rosiglitazone), respectively. The normalized activity is presented as relative activity based on the promoterless vector. Error bars represent as S.D. Data are mean ± S.D. of three independent experiments. (E) Chromatin immunoprecipitation using the livers of Hnf4af/f and Hnf4aHep mice with 4 μg of anti-PPARα, PPARβ/δ, and PPARγ antibodies and normal goat IgG. The regions between −538 and −465 containing the PPRE in the Fatp1 promoter, between −2899 and −2833 containing the PPRE in the Ehhadh promoter, between −513 and −358 containing the PPRE in the Hmgcs2 promoter, and between +45,820 and + 45,893 without an PPRE in the mouse Hmgcs2 gene were amplified, respectively. For the Ehhadh and Hmgcs2 promoters containing the PPRE, only anti-PPARα antibody was used. The data from qPCR was normalized relative to the input and expressed as -fold enrichment over data from IgG control. Error bars represent S.D. Data are mean ± S.D. of three independent experiments. ∗, P < 0.05 compared to Hnf4af/f mice.

Article Snippet: ChIP on liver samples was performed according to a protocol using 4 μg of anti-PPARα, PPARβ, PPARγ antibodies and normal mouse IgG (Santa Cruz Biotechnology).

Techniques: Binding Assay, Construct, Transfection, Expressing, Plasmid Preparation, Activity Assay, Chromatin Immunoprecipitation, Amplification, Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: TP53INP2 Promotes Bovine Adipocytes Differentiation Through Autophagy Activation

doi: 10.3390/ani9121060

Figure Lengend Snippet: List of the sequences of all primers, GenBank accession numbers and the size of the product in this study.

Article Snippet: Next, the membrane was incubated with antibodies against β-ACTIN (1:5000, NOVUS, HK, NP_776404.2), TP53INP2 (1:2000, AVIVA Systems Biology, San Diego, CA, USA, XP_003586891.1), PPARγ (1:1000, Boster, Wuhan, China, NP_851367.1), PLIN2 (1:2000, Abcam, Cambridge, UK, NP_776405.1), FASN (1:2000, Abcam, NP_777087.1), p62 (1:2000, Abcam, NP_788814.1), or LC3 (1:2000, Abcam, NP_001001169.1) for 12 h at 4 °C.

Techniques: Sequencing

Journal: Animals : an Open Access Journal from MDPI

Article Title: TP53INP2 Promotes Bovine Adipocytes Differentiation Through Autophagy Activation

doi: 10.3390/ani9121060

Figure Lengend Snippet: Effect of TP53INP2 on the differentiation of bovine adipocytes. ( a ) The mRNA expression pattern of TP53INP2 during differentiation was measured by RT-qPCR. ( b ) PPARγ , ( c ) PLIN2 and ( d ) FASN mRNA levels decreased during differentiation and were determined by RT-qPCR after knocking down of TP53INP2. ( e )The protein expression levels of PPARγ, PLIN2 and FASN decreased during differentiation and were measured by Western blot after knocking down of TP53INP2. ( f ) PPARγ , ( g ) PLIN2 and ( h ) FASN mRNA levels increased during differentiation and were determined by RT-qPCR after overexpressing of TP53INP2. ( i ) The protein expression levels of PPARγ, PLIN2 and FASN increased during differentiation and were measured by Western blot after overexpressing of TP53INP2. Bar graphs are expressed as mean ± S. E.M. ( n = 3), *, p < 0.05; **, p < 0.01, compared with NC and empty vector. NC, negative control.

Article Snippet: Next, the membrane was incubated with antibodies against β-ACTIN (1:5000, NOVUS, HK, NP_776404.2), TP53INP2 (1:2000, AVIVA Systems Biology, San Diego, CA, USA, XP_003586891.1), PPARγ (1:1000, Boster, Wuhan, China, NP_851367.1), PLIN2 (1:2000, Abcam, Cambridge, UK, NP_776405.1), FASN (1:2000, Abcam, NP_777087.1), p62 (1:2000, Abcam, NP_788814.1), or LC3 (1:2000, Abcam, NP_001001169.1) for 12 h at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: TP53INP2 Promotes Bovine Adipocytes Differentiation Through Autophagy Activation

doi: 10.3390/ani9121060

Figure Lengend Snippet: Activating the transcriptional activity of PPARγ can restore the decrease in lipid droplets and downregulation of differentiation genes caused by knocking down of TP53INP2. ( a ) PPARγ , ( b ) PLIN2 , and ( c ) FASN mRNA expression were evaluated via RT-qPCR, rosiglitazone upregulated the mRNA levels of PLIN2 caused by decreasing the expression of TP53INP2. ( d ) PPARγ, PLIN2 and FASN protein expression were evaluated via Western blot, rosiglitazone upregulated the protein level of PLIN2 and FASN caused by decreasing the expression of TP53INP2. ( e ) Lipid droplets was stained via HCS LipidTOX™ Deep Red Neutral Lipid Stain (red) (20 μm), rosiglitazone restored the reduction of lipids caused by decreasing the expression of TP53INP2. Transfected TP53INP2 siRNA and NC into bovine adipocytes, next induced differentiation 3 days. Bar graphs are expressed as mean ± S.E.M. ( n = 3), **, p < 0.01, compared with NC. NC, negative control; DAPI, 4’,6-diamidino-2-phenylindole; R, rosiglitazone.

Article Snippet: Next, the membrane was incubated with antibodies against β-ACTIN (1:5000, NOVUS, HK, NP_776404.2), TP53INP2 (1:2000, AVIVA Systems Biology, San Diego, CA, USA, XP_003586891.1), PPARγ (1:1000, Boster, Wuhan, China, NP_851367.1), PLIN2 (1:2000, Abcam, Cambridge, UK, NP_776405.1), FASN (1:2000, Abcam, NP_777087.1), p62 (1:2000, Abcam, NP_788814.1), or LC3 (1:2000, Abcam, NP_001001169.1) for 12 h at 4 °C.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Western Blot, Staining, Transfection, Negative Control

Journal: bioRxiv

Article Title: Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

doi: 10.1101/2023.10.25.563939

Figure Lengend Snippet: (A) Immunostaining of 293T cells co-transfected with plasmids encoding ZFP407 and PPARγ. (B) Subcellular fractionation of 3T3-L1 differentiated adipocytes blotted for ZFP407, PPARγ, and RXRα.

Article Snippet: Double immunofluorescent staining of cell-culture slides for ZFP407 and PPARγ was performed using anti-mouse c-Myc (9E10) (5ug/ml), (Santa Cruz Biotechnology, USA) and anti-rabbit PPAR (D69) (1:100, Cell Signaling Technology, USA) followed by incubation with Alexa-fluor ® 488 goat-anti-mouse (1:1000) and 568 goat-anti rabbit (1:1000) for 1 hour at room temperature.

Techniques: Immunostaining, Transfection, Fractionation

Journal: bioRxiv

Article Title: Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

doi: 10.1101/2023.10.25.563939

Figure Lengend Snippet: (A) Co-IP utilizing anti-RXR antibody performed following transfection of either an empty vector or co-transfection of PPARγ, ZFP407, and RXRα plasmids. (B) Co-IP of 3T3-L1 differentiated adipocyte nuclear extracts using matched IgG, anti-RXR, or anti-PPARγ antibodies.

Article Snippet: Double immunofluorescent staining of cell-culture slides for ZFP407 and PPARγ was performed using anti-mouse c-Myc (9E10) (5ug/ml), (Santa Cruz Biotechnology, USA) and anti-rabbit PPAR (D69) (1:100, Cell Signaling Technology, USA) followed by incubation with Alexa-fluor ® 488 goat-anti-mouse (1:1000) and 568 goat-anti rabbit (1:1000) for 1 hour at room temperature.

Techniques: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Cotransfection

Journal: bioRxiv

Article Title: Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

doi: 10.1101/2023.10.25.563939

Figure Lengend Snippet: (A) Overlap of DNA binding sites for ZFP407 and PPARγ determined via ChIP-Seq. (B) Co-occupancy of ZFP407 and PPARγ observed in promoter regions of GLUT4 and UBQLN1 genes, PPARγ targets downstream of the Insulin Receptor (INSR). Genomic intron/exon structure for each gene is shown below ChIP data. (C) Top 5 most significant HOMER determined enriched motifs in non-overlapping ZFP407 ChIP- Seq DNA binding sites. (D) Top 5 motifs enriched in non-overlapping PPARγ ChIP-Seq sites. (E) Top 5 motifs enriched in overlapping ChIP-Seq sites. ZFP407 data is from our ChIP-Seq assay, PPARγ data is from Haakonsson et al. (2013).

Article Snippet: Double immunofluorescent staining of cell-culture slides for ZFP407 and PPARγ was performed using anti-mouse c-Myc (9E10) (5ug/ml), (Santa Cruz Biotechnology, USA) and anti-rabbit PPAR (D69) (1:100, Cell Signaling Technology, USA) followed by incubation with Alexa-fluor ® 488 goat-anti-mouse (1:1000) and 568 goat-anti rabbit (1:1000) for 1 hour at room temperature.

Techniques: Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

doi: 10.1101/2023.10.25.563939

Figure Lengend Snippet: ZFP407 directly binds PPARγ and RXR via LxxLL or [I/L]xx[I/V]I consensus motifs and this interaction is required for transcriptional activation. Putative PPARγ and RXRα interaction motifs in ZFP407. (A) Amino acids 1984-1998 and (C) 2137-2151 of human ZFP407 are shown aligned with other species. Putative binding motifs are highlighted in yellow. (B,D) 293T cells co-transfected with the indicated plasmids and the PPRE PPARγ luciferase reporter. LxxLL and IxxII indicate ZFP407 expression vectors with the indicated motif mutated to contain a binding-disruptive alanine.

Article Snippet: Double immunofluorescent staining of cell-culture slides for ZFP407 and PPARγ was performed using anti-mouse c-Myc (9E10) (5ug/ml), (Santa Cruz Biotechnology, USA) and anti-rabbit PPAR (D69) (1:100, Cell Signaling Technology, USA) followed by incubation with Alexa-fluor ® 488 goat-anti-mouse (1:1000) and 568 goat-anti rabbit (1:1000) for 1 hour at room temperature.

Techniques: Activation Assay, Binding Assay, Transfection, Luciferase, Expressing

Journal: bioRxiv

Article Title: Molecular regulation of PPARγ/RXRα signaling by the novel cofactor ZFP407

doi: 10.1101/2023.10.25.563939

Figure Lengend Snippet: Co-transfection of differentiated adipocyte 3T3-L1 cells with ZFP407 and wild-type (WT) or indicated PPARγ mutant measured as relative fold-increase of PPRE when compared to an empty vector control.

Article Snippet: Double immunofluorescent staining of cell-culture slides for ZFP407 and PPARγ was performed using anti-mouse c-Myc (9E10) (5ug/ml), (Santa Cruz Biotechnology, USA) and anti-rabbit PPAR (D69) (1:100, Cell Signaling Technology, USA) followed by incubation with Alexa-fluor ® 488 goat-anti-mouse (1:1000) and 568 goat-anti rabbit (1:1000) for 1 hour at room temperature.

Techniques: Cotransfection, Mutagenesis, Plasmid Preparation, Control

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: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Scheme of PPARγ function and regulation of LEF1/phosphor-β-catenin and MMP2, MMP9 and calpain-2 in urothelial carcinoma. Rosiglitazone is PPARγ agonist, and GW9662 is PPARγ antagonist

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques:

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: The pathway map of PPAR signaling in patients with UC after RT was selected using the KEGG pathway analysis

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques:

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: The expression of PPARγ was examined in specimens of normal urothelium and UC after RT using immunohistochemistry (a) and RT-PCR (b). Three representative images were shown

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Sequences of primers and siRNA oligonucleotides

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Sequencing, Negative Control

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Relative mRNA levels of PPARγ (a) and RXRα (b) were detected in both normal urothelium and UC of patients with primary UC and recipients with UC after RT by quantitative RT-PCR. *p < 0.05; **p < 0.01

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Quantitative RT-PCR

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Expression of LEF1, phospho-β-catenin and total β-catenin were studied by western blot (a), and quantification of LEF1 expression (c) and phospho-β-catenin expression (d). Co-immunoprecipitation of LEF1 and phospho-β-catenin (b) and quantification of expression of LEF1 pulled by phospho-β-catenin (e). Two UC cell lines, T24 and 5637, were treated with PPARγ agonist Rosiglitazone (20 μM) or PPARγ antagonist GW9662 (20 μM). Experiments were repeated for three times. *p < 0.05

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Expression of LEF1 in T24 and 5637 treated with PBS, blank vector, negative siRNA, expression vector containing the full-length cDNA for LEF1 and siRNA targeting LEF1 by western blot(a). Cell proliferation of T24 treated with following five agents respectively, and PPARγ agonist Rosiglitazone (20 μM) or PPARγ antagonist GW9662 (20 μM) normalized by control (b). Experiments were repeated for three times. *p < 0.05

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing, Plasmid Preparation, Western Blot, Control

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: The correlation between PPARγ expression and clinicopathologic features of patients with UC after RT

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Expression of MMP2, MMP9 and calpain-2 in T24 and 5637 treated with PPARγ agonist Rosiglitazone (20 μM) or PPARγ antagonist GW9662 (20 μM) (a), and quantification of expression of MMP2 (b), MMP9 (c) and calpain-2 (d). Experiments were repeated for three times. *p < 0.05; **p < 0.01

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Expressing

Journal: Bioengineered

Article Title: Peroxisome proliferator-activated receptor γ (PPARγ) suppresses the proliferation and metastasis of patients with urothelial carcinoma after renal transplantation by inhibiting LEF1/β-catenin signaling

doi: 10.1080/21655979.2020.1843834

Figure Lengend Snippet: Migration (a) and invasion (b) of T24 treated with PBS, blank vector, negative siRNA, expression vector containing the full-length cDNA for LEF1 and siRNA targeting LEF1, followed by PPARγ agonist Rosiglitazone (20 μM) or PPARγ antagonist GW9662 (20 μM) normalized by control. Experiments were repeated for three times. *p < 0.05; **p < 0.01

Article Snippet: The PPARγ agonist Rosiglitazone, and the antagonist GW9662 were purchased from Selleck Chemicals (TX, USA).

Techniques: Migration, Plasmid Preparation, Expressing, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity

doi: 10.1038/s41392-024-01930-0

Figure Lengend Snippet: ANXA1 deficiency induces increased SMAD4 and PPARγ protein levels and promotes adipogenesis. a mRNA abundance of Anxa1 and genes closely related to lipogenesis in SVFs from WT and Anxa1 Tg mice ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. b mRNA abundance of Ppara , Pparg and Ppard in SVFs from WT and Anxa1 Tg mice ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. c Representative western blot of ANXA1 at different time points after adipogenic induction during the adipogenesis of SVFs ( n = 3 per group). d Fold change of ANXA1 protein levels, quantified from c ( n = 3 per group). Student’s t test was used for analysis. e mRNA levels of Anxa1 at different time points after adipogenic induction during the adipogenesis of SVFs ( n = 6 per group). Student’s t test was used for analysis. f Oil Red O staining of SVFs at day 0, day 2, day 5 and day 8 after adipogenic induction from WT and Anxa1 Tg mice ( n = 6 per group). Scale bar: 800 µm. g Oil Red O staining (left) was quantified as Oil Red O-positive area (right) in SVFs induced to differentiate on day 2 from Anxa1 fl/fl and Anxa1 AKO mice ( n = 6 per group). Student’s t test was used for analysis. Scale bar: 400 µm. h mRNA abundance of genes closely related to adipogenesis in SVFs from WT and Anxa1 Tg mice ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. i mRNA abundance of genes closely related to adipogenesis in SVFs from Anxa1 fl/fl and Anxa1 AKO mice ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. j Representative western blotting and quantification of SMAD4 and PPARγ from SVFs transfected with ANXA1 siRNA or negative control (NC) siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. k mRNA abundance of Pparg and genes closely related to adipogenesis in SVFs transfected with ANXA1 siRNA or NC siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. l Representative western blotting and quantification of SMAD1, SMAD4, and SMAD5 in SVFs from WT and Anxa1 Tg mice ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. m Representative western blotting and quantification of ANXA1 and SMAD4 from SVFs at different time points after transfection with ANXA1 siRNA and ANXA1 siRNA(1) ( n = 3–4 per group). n Spearman correlation between fold change of ANXA1 and SMAD4 protein levels ( n = 3–4 per group). o mRNA abundance of Smad4 , Pparg and genes closely related to adipogenesis in SVFs transfected with SMAD4 siRNA or NC siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. p mRNA abundance of Pparg and genes closely related to adipogenesis in SVFs transfected with SMAD4 adenovirus (Silencing SMAD4) or NC adenovirus for 72 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. q After transfected with SMAD4 adenovirus or NC adenovirus for 72 h, Oil Red O staining of SVFs at different time points after adipogenic induction ( n = 6 per group). Scale bar: 800 µm. r mRNA abundance of Pparg and genes closely related to adipogenesis in SVFs transfected with SMAD4 lentivirus (Overexpressing SMAD4) or NC lentivirus for 96 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. s After transfected with SMAD4 lentivirus or NC lentivirus for 96 h, Oil Red O staining of SVFs at different time points after adipogenic induction ( n = 6 per group). Scale bar: 800 µm. t Predicted SMAD4 binding Pparg promoter motifs by UCSC Genome Browser Home and PROMO. u The binding of Pparg promoter motifs and IgG, Histone H3 and SMAD4 was further explored by ChIP-PCR assays in SVFs from WT , Anxa1 Tg , Anxa1 fl/fl and Anxa1 AKO . v – w The binding of Pparg promoter region and IgG, Histone H3 and SMAD4 was further explored by ChIP-qPCR assays in SVFs from WT , Anxa1 Tg , Anxa1 fl/fl and Anxa1 AKO . x Schematic diagram of the ANXA1-SMAD4-PPARγ-Adipogenesis axis

Article Snippet: The membranes were blocked in 5% skim milk and incubated at room temperature for 1 h. They were then incubated with primary antibodies against GAPDH (Proteintech, 60004-1-Ig, 1:5000), β-actin (Beyotime, AF0003, 1:1000), ANXA1 (Abcam, ab214486, 1:3000), ADIPOQ (Proteintech, 66239-1-Ig, 1:1000), SMAD4 (Proteintech, 10231-1-AP, 1:1000), P53 (Proteintech, 10442-1-AP, 1:5000), PPARγ (Proteintech, 16643-1-AP, 1:2000), PDLIM7 (Proteintech, 10221-1-AP, 1:1000), MYCBP2 (Millipore MABN2397, 1:500), Ubiquitin (P37) (Cell Signaling, 58395 1:1000), K48-linkage specific polyubiquitin (Cell Signaling, 4289, 1:1000), K63-linkage specific polyubiquitin (D7A11) (Cell Signaling, 5621, 1:1000), DYKDDDDK tag (Proteintech, 20543-1-AP, 1:2000), and HA tag (Proteintech, 51064-2-AP, 1:5000) for 12 h at 4 °C.

Techniques: Western Blot, Staining, Transfection, Negative Control, Binding Assay, ChIP-qPCR

Journal: Signal Transduction and Targeted Therapy

Article Title: Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity

doi: 10.1038/s41392-024-01930-0

Figure Lengend Snippet: The interaction between ANXA1 and PDLIM7 increases the degradation of SMAD4 after ubiquitination and inhibits adipogenesis in SVFs. a mRNA abundance of Smad4 in SVFs transfected with ANXA1 siRNA or NC siRNA for 48 h ( n = 6 per group). Student’s t test was used for analysis. b Representative western blotting and quantification of SMAD4 from WT -SVFs, Anxa1 Tg -SVFs, and WT -SVFs and Anxa1 Tg -SVFs incubated with 10 µM MG132 for 6 h ( n = 3 per group). Student’s t test was used for analysis. c SVFs were transfected with ANXA1 siRNA, ANXA1 siRNA(1) or NC siRNA for 48 h, followed by incubation with 10 µM MG132 for 6 h. Cell lysates were immunoprecipitated with an anti-SMAD4 antibody and then immunoblotted with an anti-ubiquitin (Ub) antibody, anti-K48-linkage specific polyubiquitin (K 48 -Ub) antibody and anti-K63-linkage specific polyubiquitin (K 63 -Ub) antibody. d The polar coordinate bar chart shows that PDLIM7 has a strong interaction with ANXA1 in the IP-MS results for ANXA1 and IgG. e Co-immunoprecipitation assay of ANXA1 and PDLIM7 in SVFs. f Representative western blotting and quantification of SMAD4 and PDLIM7 from SVFs transfected with PDLIM7 siRNA or NC siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. g mRNA abundance of Pdlim7 , Pparg and genes closely related to adipogenesis in SVFs transfected with PDLIM7 siRNA or NC siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. h After incubation with PDLIM7 lentivirus (Overexpressing PDLIM7) or NC lentivirus for 96 h, Oil Red O staining of SVFs at different time points after adipogenic induction ( n = 6 per group). Scale bar: 800 µm. i SVFs were transfected with PDLIM7 siRNA, PDLIM7 siRNA(1) or NC siRNA for 48 h, followed by incubation with 10 µM MG132 for 6 h. Cell lysates were immunoprecipitated with an anti-SMAD4 antibody and then immunoblotted with an anti-Ub antibody, anti-K 48 -Ub antibody and anti-K 63 -Ub antibody. j Schematic diagram of peptide segment design. k HEK 293T cells were transfected with different peptide segment vector plasmids or empty plasmids (Puc57) for 48 h. Cell lysates were immunoprecipitated with an anti-Flag tag antibody and then immunoblotted with an anti-HA tag antibody, or conversely, cell lysates were immunoprecipitated with an anti-HA tag antibody and then immunoblotted with an anti-Flag tag antibody. l Schematic representation of the interaction of ANXA1 with PDLIM7. ANXA1-pfam1 in yellow, ANXA1-pfam2 in blue, PDLIM7-pfam1-3 in green and PDLIM7-pfam4 in orange-red. m Schematic diagram of the ANXA1-PDLIM7-SMAD4-PPARγ-Adipogenesis axis

Article Snippet: The membranes were blocked in 5% skim milk and incubated at room temperature for 1 h. They were then incubated with primary antibodies against GAPDH (Proteintech, 60004-1-Ig, 1:5000), β-actin (Beyotime, AF0003, 1:1000), ANXA1 (Abcam, ab214486, 1:3000), ADIPOQ (Proteintech, 66239-1-Ig, 1:1000), SMAD4 (Proteintech, 10231-1-AP, 1:1000), P53 (Proteintech, 10442-1-AP, 1:5000), PPARγ (Proteintech, 16643-1-AP, 1:2000), PDLIM7 (Proteintech, 10221-1-AP, 1:1000), MYCBP2 (Millipore MABN2397, 1:500), Ubiquitin (P37) (Cell Signaling, 58395 1:1000), K48-linkage specific polyubiquitin (Cell Signaling, 4289, 1:1000), K63-linkage specific polyubiquitin (D7A11) (Cell Signaling, 5621, 1:1000), DYKDDDDK tag (Proteintech, 20543-1-AP, 1:2000), and HA tag (Proteintech, 51064-2-AP, 1:5000) for 12 h at 4 °C.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Incubation, Immunoprecipitation, Protein-Protein interactions, Co-Immunoprecipitation Assay, Staining, Plasmid Preparation, FLAG-tag

Journal: Signal Transduction and Targeted Therapy

Article Title: Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity

doi: 10.1038/s41392-024-01930-0

Figure Lengend Snippet: The interaction between PDLIM7 and MYCBP2 inhibits MYCBP2-mediated ubiquitination of SMAD4 and promotes adipogenesis in SVFs. a The polar coordinate bar chart shows that MYCBP2 has a strong interaction with PDLIM7 in the IP-MS results for PDLIM7 and IgG. b Co-immunoprecipitation assay of PDLIM7 and MYCBP2 in SVFs. c Representative western blotting and quantification of MYCBP2, PPARγ, SMAD4 and PDLIM7 from SVFs transfected with MYCBP2 siRNA or NC siRNA for 48 hs ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. d mRNA abundance of Anxa1 , Pdlim7 , Mycbp2 and Smad4 in SVFs transfected with MYCBP2 siRNA or NC siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. e mRNA abundance of Pparg and genes closely related to adipogenesis in SVFs transfected with MYCBP2 siRNA or NC siRNA for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. f Co-immunoprecipitation assay of MYCBP2 and SMAD4 in SVFs. g SVFs were transfected with MYCBP2 siRNA, MYCBP2 siRNA(1) or NC siRNA for 48 h, followed by incubation with 10 µM MG132 for 6 h. Cell lysates were immunoprecipitated with an anti-SMAD4 antibody and then immunoblotted with an anti-Ub antibody, anti-K 48 -Ub antibody and anti-K 63 -Ub antibody. h After transfected with MYCBP2 adenovirus (Silencing MYCBP2) or NC adenovirus for 72 h, Oil Red O staining of SVFs at different time points after adipogenic induction ( n = 6 per group). Scale bar: 800 µm. i mRNA abundance of Anxa1 , Pdlim7 , Mycbp2 , Smad4 , Pparg and genes closely related to adipogenesis in SVFs transfected with MYCBP2 adenovirus or NC adenovirus for 72 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. Representative western blotting ( j ) and quantification ( k ) of MYCBP2, SMAD4, PPARγ and ANXA1 from SVFs transfected with MYCBP2 adenovirus or NC adenovirus for 96 h and transfected with ANXA1 siRNA or NC siRNA for 48 h ( n = 3 per group). One-way ANOVA and Dunn post hoc test were used for analysis. l Co-immunoprecipitation assay of MYCBP2 and PDLIM7 in SVFs transfected with ANXA1 siRNA or NC siRNA for 48 h followed by incubation with 10 µM MG132 for 6 h. m Co-immunoprecipitation assay of MYCBP2 and SMAD4 in SVFs transfected with ANXA1 siRNA or NC siRNA for 48 h followed by incubation with 10 µM MG132 for 6 h. n Co-immunoprecipitation assay of MYCBP2 and SMAD4 in SVFs transfected with PDLIM7 siRNA or NC siRNA for 48 h followed by incubation with 10 µM MG132 for 6 h. o Schematic diagram of peptide segment design. p HEK 293T cells were transfected with different peptide segment vector plasmids or empty plasmids for 48 h. Cell lysates were immunoprecipitated with an anti-Flag tag antibody and then immunoblotted with an anti-HA tag antibody, or conversely, cell lysates were immunoprecipitated with an anti-HA tag antibody and then immunoblotted with an anti-Flag tag antibody. q Schematic representation of the interaction of PDLIM7 with MYCBP2. PDLIM7-pfam1-3 in green, PDLIM7-pfam4 in orange-red, RING in yellow and MYCBP2 in gray except for RING. r Schematic representation of the interaction of MYCBP2 with SMAD4. RING in yellow, MYCBP2 in gray except for RING and SMAD4 in orange-red. s Schematic diagram of the ANXA1-PDLIM7-MYCBP2-SMAD4-PPARγ-Adipogenesis axis

Article Snippet: The membranes were blocked in 5% skim milk and incubated at room temperature for 1 h. They were then incubated with primary antibodies against GAPDH (Proteintech, 60004-1-Ig, 1:5000), β-actin (Beyotime, AF0003, 1:1000), ANXA1 (Abcam, ab214486, 1:3000), ADIPOQ (Proteintech, 66239-1-Ig, 1:1000), SMAD4 (Proteintech, 10231-1-AP, 1:1000), P53 (Proteintech, 10442-1-AP, 1:5000), PPARγ (Proteintech, 16643-1-AP, 1:2000), PDLIM7 (Proteintech, 10221-1-AP, 1:1000), MYCBP2 (Millipore MABN2397, 1:500), Ubiquitin (P37) (Cell Signaling, 58395 1:1000), K48-linkage specific polyubiquitin (Cell Signaling, 4289, 1:1000), K63-linkage specific polyubiquitin (D7A11) (Cell Signaling, 5621, 1:1000), DYKDDDDK tag (Proteintech, 20543-1-AP, 1:2000), and HA tag (Proteintech, 51064-2-AP, 1:5000) for 12 h at 4 °C.

Techniques: Ubiquitin Proteomics, Protein-Protein interactions, Co-Immunoprecipitation Assay, Western Blot, Transfection, Incubation, Immunoprecipitation, Staining, Plasmid Preparation, FLAG-tag

Journal: Signal Transduction and Targeted Therapy

Article Title: Annexin A1 binds PDZ and LIM domain 7 to inhibit adipogenesis and prevent obesity

doi: 10.1038/s41392-024-01930-0

Figure Lengend Snippet: Ac2-26 inhibits adipogenesis and prevents obesity in HFD mice. a Representative western blotting and quantification of SMAD4 from SVFs incubated with 0.1 mg/ml Ac2-26 or DMSO (NC) for 48 h ( n = 6 per group). Student’s t test was used for analysis. b mRNA abundance of Pparg and genes closely related to adipogenesis in SVFs incubated with 0.1 mg/ml Ac2-26 or DMSO for 48 h ( n = 6 per group). One-way ANOVA and Dunn post hoc test were used for analysis. c Oil Red O staining of SVFs at different time points after adipogenic induction incubated with 0.1 mg/ml Ac2-26 or DMSO ( n = 6 per group). Scale bar: 800 µm. d ICC experiments demonstrate the interaction between PDLIM7 and FITC- Ac2-26. Scale bar: 800 µm, 160 µm. e – j Eight-week-old db/m mice, db/db mice, and db/db mice were injected intraperitoneally with 0, 0.5, 1.0, or 2.0 mg/kg of Ac2-26 (dissolved in PBS) for 10 weeks and were fed with HFD throughout. e Body weight of the five groups of the mice ( n = 8 per group). Student’s t test was used for analysis. f – h Blood glucose, total cholesterol (TC), and triglyceride (TG) concentrations in db/m mice, db/db mice, and db/db mice injected intraperitoneally with 0.0 or 2.0 mg/kg of Ac2-26 ( n = 5–9 per group). Student’s t test was used for analysis. i Results of glucose tolerance test (GTT)(left) were quantified as area under the curve (AUC)(right) for db/m mice, db/db mice, and db/db mice injected intraperitoneally with 0.0 or 2.0 mg/kg of Ac2-26 ( n = 7 per group). One-way ANOVA and Dunn post hoc test were used for analysis. j Results of insulin tolerance test (ITT)(left) were quantified as AUC(right) for the three groups of mice ( n = 7 per group). One-way ANOVA and Dunn post hoc test were used for analysis. k Schematic created with BioRender.com to better illustrate the conceptual advancement in our understanding of how ANXA1 affects adipogenesis. Overexpression of ANXA1 in SVFs enhanced its interaction with PDLIM7, thereby weakening the interaction of PDLIM7 with MYCBP2.This exposed the MYCBP2-binding site, allowing it to bind more readily to SMAD4 and mediate its ubiquitination and degradation. SMAD4 degradation downregulated PPARγ transcription and reduced adipogenesis. Conversely, knocking out ANXA1 made HFD-mice more obese

Article Snippet: The membranes were blocked in 5% skim milk and incubated at room temperature for 1 h. They were then incubated with primary antibodies against GAPDH (Proteintech, 60004-1-Ig, 1:5000), β-actin (Beyotime, AF0003, 1:1000), ANXA1 (Abcam, ab214486, 1:3000), ADIPOQ (Proteintech, 66239-1-Ig, 1:1000), SMAD4 (Proteintech, 10231-1-AP, 1:1000), P53 (Proteintech, 10442-1-AP, 1:5000), PPARγ (Proteintech, 16643-1-AP, 1:2000), PDLIM7 (Proteintech, 10221-1-AP, 1:1000), MYCBP2 (Millipore MABN2397, 1:500), Ubiquitin (P37) (Cell Signaling, 58395 1:1000), K48-linkage specific polyubiquitin (Cell Signaling, 4289, 1:1000), K63-linkage specific polyubiquitin (D7A11) (Cell Signaling, 5621, 1:1000), DYKDDDDK tag (Proteintech, 20543-1-AP, 1:2000), and HA tag (Proteintech, 51064-2-AP, 1:5000) for 12 h at 4 °C.

Techniques: Western Blot, Incubation, Staining, Injection, Over Expression, Binding Assay, Ubiquitin Proteomics

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Fatty Acids Regulate Endothelial Lipase and Inflammatory Markers in Macrophages and in Mouse Aorta

doi: 10.1161/atvbaha.112.300188

Figure Lengend Snippet: Figure 4. Interactions of eicosapentaenoic acid (EPA) and palmitic acid (PA) with a peroxisome proliferator activated receptor (PPAR)γ agonist/antagonist on endothelial lipase (EL) expression in macrophages. J774 (A–C) or peritoneal macrophages (D–E) were pretreated with or without fatty acids for 3 h and then continuously cultured with PPARγ agonist rosiglitazone (ROSI) and/or PPARγ antagonist GW9662 for 4 h (mRNA) or 21 h (protein). *P<0.05, **P<0.01 (Student’s t test).

Article Snippet: For experiments requiring PPARγ mRNA knock-down, J774 cells (50–70% confluence) were transfected with optimized concentrations of either mouse PPARγ short hairpin RNA (shRNA) plasmid (Santa Cruz Biotechnology, Inc), or control nonsense shRNA plasmid using shRNA transfection reagent (sc-29528), according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture