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