Structured Review

Addgene inc padcmv v5 dest s727d stat3 3xflag
a . Il6 expression in 12-week high fat diet (HFD) fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control for 30 minutes before sacrifice and tissue collection. Expression levels normalized to vehicle within each tissue. Individual data points plotted ± SEM ( n = 12 mice per treatment group). p value = 0.0003 (iWAT) and 0.006 (eWAT) b . Serum IL-6 levels in 12-week HFD fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control before sacrifice and blood collection. Data are represented as mean ± SEM ( n = 3 mice per genotype at each time point). p value < 0.0001 WT vs Il6 KO. Quantification of phosphorylation of c . HSL at serine 563 over total HSL ( p value < 0.0001 20 min vs. basal) d . p38 at threonine 180 and tyrosine 182 over total p38 ( p value = 0.003 20 min vs. basal) e . <t>STAT3</t> at tyrosine 705 over total STAT3 ( p value < 0.0001 120 min vs. basal) and f . STAT3 at serine 727 over total STAT3 ( p value = 0.003 20 min vs. basal). c .- f . Western blots shown in . Individual data points plotted ± SEM ( n = 3 WT, 4 SAKO mice per time point). * p value < 0.05 from post hoc analysis after significant two-way ANOVA.
Padcmv V5 Dest S727d Stat3 3xflag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/padcmv v5 dest s727d stat3 3xflag/product/Addgene inc
Average 91 stars, based on 1 article reviews
Price from $9.99 to $1999.99
padcmv v5 dest s727d stat3 3xflag - by Bioz Stars, 2024-07
91/100 stars

Images

1) Product Images from "Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3"

Article Title: Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3

Journal: Nature metabolism

doi: 10.1038/s42255-020-0217-6

a . Il6 expression in 12-week high fat diet (HFD) fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control for 30 minutes before sacrifice and tissue collection. Expression levels normalized to vehicle within each tissue. Individual data points plotted ± SEM ( n = 12 mice per treatment group). p value = 0.0003 (iWAT) and 0.006 (eWAT) b . Serum IL-6 levels in 12-week HFD fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control before sacrifice and blood collection. Data are represented as mean ± SEM ( n = 3 mice per genotype at each time point). p value < 0.0001 WT vs Il6 KO. Quantification of phosphorylation of c . HSL at serine 563 over total HSL ( p value < 0.0001 20 min vs. basal) d . p38 at threonine 180 and tyrosine 182 over total p38 ( p value = 0.003 20 min vs. basal) e . STAT3 at tyrosine 705 over total STAT3 ( p value < 0.0001 120 min vs. basal) and f . STAT3 at serine 727 over total STAT3 ( p value = 0.003 20 min vs. basal). c .- f . Western blots shown in . Individual data points plotted ± SEM ( n = 3 WT, 4 SAKO mice per time point). * p value < 0.05 from post hoc analysis after significant two-way ANOVA.
Figure Legend Snippet: a . Il6 expression in 12-week high fat diet (HFD) fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control for 30 minutes before sacrifice and tissue collection. Expression levels normalized to vehicle within each tissue. Individual data points plotted ± SEM ( n = 12 mice per treatment group). p value = 0.0003 (iWAT) and 0.006 (eWAT) b . Serum IL-6 levels in 12-week HFD fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control before sacrifice and blood collection. Data are represented as mean ± SEM ( n = 3 mice per genotype at each time point). p value < 0.0001 WT vs Il6 KO. Quantification of phosphorylation of c . HSL at serine 563 over total HSL ( p value < 0.0001 20 min vs. basal) d . p38 at threonine 180 and tyrosine 182 over total p38 ( p value = 0.003 20 min vs. basal) e . STAT3 at tyrosine 705 over total STAT3 ( p value < 0.0001 120 min vs. basal) and f . STAT3 at serine 727 over total STAT3 ( p value = 0.003 20 min vs. basal). c .- f . Western blots shown in . Individual data points plotted ± SEM ( n = 3 WT, 4 SAKO mice per time point). * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

Techniques Used: Expressing, Western Blot

a ., f .- h . Western blot analysis of STAT3 phosphorylation in fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243; HSL phosphorylation in the lipid droplet fraction also shown to demonstrate effectiveness of CL-316,243 treatment. b . Western blot analysis of STAT3 phosphorylation in fractionated adipose tissue collected after intraperitoneal injection of 1 mg/kg CL-316,243 (20 or 120 min) or vehicle control (20 min after vehicle injection). c . Confocal images of PPDIVs stained with Bodipy, DAPI, STAT3 (Alex fluor 555 conjugated secondary) and pSer 727 STAT3 (Alex fluor 647 conjugated secondary). Individual channels of two representative vehicle and CL treated cells shown. Scale bar = 10 μm. d . 3-D representation of z-stack images of PPDIVs stained with bodipy (green), Dapi (blue) STAT3 (yellow) or pSer 727 STAT3 (red) are shown with colocalization of Bodipy and STAT3 shown in pink, and colocalization of Bodipy pSer 727 STAT3 shown in white. e . Quantification of colocalization of STAT3 and pSer 727 STAT3 with bodipy, normalized to lipid droplet volume (top) or lipid droplet surface area (bottom). Data are represented as mean ± SEM ( n = 61 V and 64 CL lipids droplets) p value < 0.0001 WT versus SAKO pSer 727 . f . Cells pretreated with H89 (PKA inhibitor) or JAK inhibitor I for 30 min, before Cl-316,243 treatment for 60 min. g . Cells pretreated with H89, SB-303,580 or atglistatin for 30 min, before Cl-316,243 treatment for 15 min. Right panel : Quantification of Ser 727 STAT3 phosphorylation in three independent experiments, data are represented as mean ± SEM. p value < 0.0001 DMSO v vs CL, CL DMSO vs. PKAi/ATGLi and = 0,003 p38i V vs CL. h . Cl-316,243 treatment performed in the presence or absence of 2% BSA in the media. Blots are representative of results from three independent experiments, 75 kDa and 100 kDa protein marker locations indicated in pink and blue respectively. Results in c - e were replicated in an independent experiment. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.
Figure Legend Snippet: a ., f .- h . Western blot analysis of STAT3 phosphorylation in fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243; HSL phosphorylation in the lipid droplet fraction also shown to demonstrate effectiveness of CL-316,243 treatment. b . Western blot analysis of STAT3 phosphorylation in fractionated adipose tissue collected after intraperitoneal injection of 1 mg/kg CL-316,243 (20 or 120 min) or vehicle control (20 min after vehicle injection). c . Confocal images of PPDIVs stained with Bodipy, DAPI, STAT3 (Alex fluor 555 conjugated secondary) and pSer 727 STAT3 (Alex fluor 647 conjugated secondary). Individual channels of two representative vehicle and CL treated cells shown. Scale bar = 10 μm. d . 3-D representation of z-stack images of PPDIVs stained with bodipy (green), Dapi (blue) STAT3 (yellow) or pSer 727 STAT3 (red) are shown with colocalization of Bodipy and STAT3 shown in pink, and colocalization of Bodipy pSer 727 STAT3 shown in white. e . Quantification of colocalization of STAT3 and pSer 727 STAT3 with bodipy, normalized to lipid droplet volume (top) or lipid droplet surface area (bottom). Data are represented as mean ± SEM ( n = 61 V and 64 CL lipids droplets) p value < 0.0001 WT versus SAKO pSer 727 . f . Cells pretreated with H89 (PKA inhibitor) or JAK inhibitor I for 30 min, before Cl-316,243 treatment for 60 min. g . Cells pretreated with H89, SB-303,580 or atglistatin for 30 min, before Cl-316,243 treatment for 15 min. Right panel : Quantification of Ser 727 STAT3 phosphorylation in three independent experiments, data are represented as mean ± SEM. p value < 0.0001 DMSO v vs CL, CL DMSO vs. PKAi/ATGLi and = 0,003 p38i V vs CL. h . Cl-316,243 treatment performed in the presence or absence of 2% BSA in the media. Blots are representative of results from three independent experiments, 75 kDa and 100 kDa protein marker locations indicated in pink and blue respectively. Results in c - e were replicated in an independent experiment. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

Techniques Used: Western Blot, Injection, Staining, Marker

a . Left panel: Western blot of mature adipocytes isolated from 12-week old WT and SAKO mice. Right panel: Quantification of STAT3 protein relative to RalA loading control. Individual data points plotted ± SEM ( n = 3 iWAT, 2 eWAT). b . Body weight of 12-week old ND fed WT and SAKO mice. Individual data points plotted ± SEM ( n = 6 per genotype). c . Oxygen consumption rate in ND fed WT and SAKO mice at 16-weeks of age. Data are represented as mean ± SEM ( n = 16). d . Adipocyte size distribution from ND-fed 12-week old WT and SAKO eWAT ( n = 2 WT and 3 SAKO). e . Adipocyte size distribution from ND-fed 12-week old WT and SAKO iWAT ( n = 2 WT and 3 SAKO). f . Body composition of ND fed WT and SAKO mice at 12 weeks of age. Individual data points plotted ± SEM ( n =6 WT and 4 SAKO).
Figure Legend Snippet: a . Left panel: Western blot of mature adipocytes isolated from 12-week old WT and SAKO mice. Right panel: Quantification of STAT3 protein relative to RalA loading control. Individual data points plotted ± SEM ( n = 3 iWAT, 2 eWAT). b . Body weight of 12-week old ND fed WT and SAKO mice. Individual data points plotted ± SEM ( n = 6 per genotype). c . Oxygen consumption rate in ND fed WT and SAKO mice at 16-weeks of age. Data are represented as mean ± SEM ( n = 16). d . Adipocyte size distribution from ND-fed 12-week old WT and SAKO eWAT ( n = 2 WT and 3 SAKO). e . Adipocyte size distribution from ND-fed 12-week old WT and SAKO iWAT ( n = 2 WT and 3 SAKO). f . Body composition of ND fed WT and SAKO mice at 12 weeks of age. Individual data points plotted ± SEM ( n =6 WT and 4 SAKO).

Techniques Used: Western Blot, Isolation

a . Western blot analysis of fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243 or vehicle control for 60 min. b .- d . and f . Western blot analysis of input, flow through and immunoprecipitation using Myc-antibody coated beads ( b , c ) or Flag-antibody coated beads ( d , f ) of HEK293T cell lysates overexpressing Flag-tagged STAT3 and/or Myc-tagged GPAT3/GPAT4. Blots are representative of three independent replicates. Dark exposure (D.E.). e . Western blot analysis of input and immunoprecipitation using GPAT3 antibody in 3T3-L1 differentiated adipocytes treated with 10 μM CL-316,243 or vehicle control for 15 min. f . Western blot analysis of input, flow through and immunoprecipitation using Flag antibody coated beads of HEK293T cell lysates overexpressing Flag-tagged STAT3 (WT/S 727 A/S 727 D) and/or Myc-tagged GPAT3. Blots are representative of three independent replicates. Arrow indicates expected size of Ser 727 phosphorylated STAT3; the band observed in the IP samples is a larger non-specific band. g . Western blot analysis of input, flow through, and immunoprecipitation using Flag antibody coated beads from 3T3-L1 differentiated adipocytes with lentiviral overexpression of flag-tagged STAT3 (WT/Y 705 F/S 727 A) and/or Myc-tagged GPAT3, cells treated with 10 μM CL-316,243 or vehicle control for 60 min before harvest and IP. These experiments were repeated independently twice with similar results.
Figure Legend Snippet: a . Western blot analysis of fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243 or vehicle control for 60 min. b .- d . and f . Western blot analysis of input, flow through and immunoprecipitation using Myc-antibody coated beads ( b , c ) or Flag-antibody coated beads ( d , f ) of HEK293T cell lysates overexpressing Flag-tagged STAT3 and/or Myc-tagged GPAT3/GPAT4. Blots are representative of three independent replicates. Dark exposure (D.E.). e . Western blot analysis of input and immunoprecipitation using GPAT3 antibody in 3T3-L1 differentiated adipocytes treated with 10 μM CL-316,243 or vehicle control for 15 min. f . Western blot analysis of input, flow through and immunoprecipitation using Flag antibody coated beads of HEK293T cell lysates overexpressing Flag-tagged STAT3 (WT/S 727 A/S 727 D) and/or Myc-tagged GPAT3. Blots are representative of three independent replicates. Arrow indicates expected size of Ser 727 phosphorylated STAT3; the band observed in the IP samples is a larger non-specific band. g . Western blot analysis of input, flow through, and immunoprecipitation using Flag antibody coated beads from 3T3-L1 differentiated adipocytes with lentiviral overexpression of flag-tagged STAT3 (WT/Y 705 F/S 727 A) and/or Myc-tagged GPAT3, cells treated with 10 μM CL-316,243 or vehicle control for 60 min before harvest and IP. These experiments were repeated independently twice with similar results.

Techniques Used: Western Blot, Immunoprecipitation, Over Expression

a . Incorporation of 14 C-palmitic acid into triglycerides by SAKO PPDIV relative to WT control cells ± 1 μM CL-316,243. Individual data points plotted ± SEM ( n = 3 wells per condition, p values = 0.0008). b . GPAT activity in iWAT homogenates from WT and SAKO mice treated with 1 mg/kg CL-316,243 or vehicle control for 20 minutes. Individual data points plotted ± SEM ( n = 3 mice per condition, p value =0.002 WT V vs. CL, 0.019 WT vs. SAKO CL). c . Western blot analysis of GPAT3 and GPAT4 protein levels in lysates from WT and SAKO iWAT and eWAT. Results are representative of three independent experiments, 50, 75 and 100 kDa protein markers indicated in green, pink and blue respectively. d . OCR at 30 min ± 10 μM CL-316,243 in 3T3-L1 adipocytes ± JAK inhibitor I pretreatment for 30 min, normalized to baseline. Individual data points plotted ± SEM ( n = 8 wells per condition, p values < 0.0001). e . and f . OCR at 30 min ± 0.5 μM CL-316,243 in WT and SAKO PPDIV with lentiviral STAT3 (WT, S 727 A or S 727 D) or GFP overexpression. e . Data are represented as mean ± SEM ( n = 12 wells per over expression construct in each genotype, p value < 0.0001 V vs. CL, 0.002 WT CL vs. SAKO CL GFP, and 0.046 WT CL vs. SAKO CL S727A ). f . Individual data points plotted ± SEM ( n = 4 wells per condition, p value = 0.029 V vs. CL STAT3, 0.016 V vs. CL 727D, 0.047 GFP vs. STAT3 CL, 0.011 727A vs. STAT3 CL, and 0.024 727A vs. 727D CL). g . Model of FA handling in the fed state (blue arrows) versus the fasted stated (red arrows) and the signaling regulating lipolysis-driven oxidative metabolism (black arrows) in the fasted state. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.
Figure Legend Snippet: a . Incorporation of 14 C-palmitic acid into triglycerides by SAKO PPDIV relative to WT control cells ± 1 μM CL-316,243. Individual data points plotted ± SEM ( n = 3 wells per condition, p values = 0.0008). b . GPAT activity in iWAT homogenates from WT and SAKO mice treated with 1 mg/kg CL-316,243 or vehicle control for 20 minutes. Individual data points plotted ± SEM ( n = 3 mice per condition, p value =0.002 WT V vs. CL, 0.019 WT vs. SAKO CL). c . Western blot analysis of GPAT3 and GPAT4 protein levels in lysates from WT and SAKO iWAT and eWAT. Results are representative of three independent experiments, 50, 75 and 100 kDa protein markers indicated in green, pink and blue respectively. d . OCR at 30 min ± 10 μM CL-316,243 in 3T3-L1 adipocytes ± JAK inhibitor I pretreatment for 30 min, normalized to baseline. Individual data points plotted ± SEM ( n = 8 wells per condition, p values < 0.0001). e . and f . OCR at 30 min ± 0.5 μM CL-316,243 in WT and SAKO PPDIV with lentiviral STAT3 (WT, S 727 A or S 727 D) or GFP overexpression. e . Data are represented as mean ± SEM ( n = 12 wells per over expression construct in each genotype, p value < 0.0001 V vs. CL, 0.002 WT CL vs. SAKO CL GFP, and 0.046 WT CL vs. SAKO CL S727A ). f . Individual data points plotted ± SEM ( n = 4 wells per condition, p value = 0.029 V vs. CL STAT3, 0.016 V vs. CL 727D, 0.047 GFP vs. STAT3 CL, 0.011 727A vs. STAT3 CL, and 0.024 727A vs. 727D CL). g . Model of FA handling in the fed state (blue arrows) versus the fasted stated (red arrows) and the signaling regulating lipolysis-driven oxidative metabolism (black arrows) in the fasted state. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

Techniques Used: Activity Assay, Western Blot, Over Expression, Construct

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 91
    Addgene inc padcmv v5 dest s727d stat3 3xflag
    a . Il6 expression in 12-week high fat diet (HFD) fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control for 30 minutes before sacrifice and tissue collection. Expression levels normalized to vehicle within each tissue. Individual data points plotted ± SEM ( n = 12 mice per treatment group). p value = 0.0003 (iWAT) and 0.006 (eWAT) b . Serum IL-6 levels in 12-week HFD fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control before sacrifice and blood collection. Data are represented as mean ± SEM ( n = 3 mice per genotype at each time point). p value < 0.0001 WT vs Il6 KO. Quantification of phosphorylation of c . HSL at serine 563 over total HSL ( p value < 0.0001 20 min vs. basal) d . p38 at threonine 180 and tyrosine 182 over total p38 ( p value = 0.003 20 min vs. basal) e . <t>STAT3</t> at tyrosine 705 over total STAT3 ( p value < 0.0001 120 min vs. basal) and f . STAT3 at serine 727 over total STAT3 ( p value = 0.003 20 min vs. basal). c .- f . Western blots shown in . Individual data points plotted ± SEM ( n = 3 WT, 4 SAKO mice per time point). * p value < 0.05 from post hoc analysis after significant two-way ANOVA.
    Padcmv V5 Dest S727d Stat3 3xflag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/padcmv v5 dest s727d stat3 3xflag/product/Addgene inc
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    padcmv v5 dest s727d stat3 3xflag - by Bioz Stars, 2024-07
    91/100 stars
      Buy from Supplier

    Image Search Results


    a . Il6 expression in 12-week high fat diet (HFD) fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control for 30 minutes before sacrifice and tissue collection. Expression levels normalized to vehicle within each tissue. Individual data points plotted ± SEM ( n = 12 mice per treatment group). p value = 0.0003 (iWAT) and 0.006 (eWAT) b . Serum IL-6 levels in 12-week HFD fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control before sacrifice and blood collection. Data are represented as mean ± SEM ( n = 3 mice per genotype at each time point). p value < 0.0001 WT vs Il6 KO. Quantification of phosphorylation of c . HSL at serine 563 over total HSL ( p value < 0.0001 20 min vs. basal) d . p38 at threonine 180 and tyrosine 182 over total p38 ( p value = 0.003 20 min vs. basal) e . STAT3 at tyrosine 705 over total STAT3 ( p value < 0.0001 120 min vs. basal) and f . STAT3 at serine 727 over total STAT3 ( p value = 0.003 20 min vs. basal). c .- f . Western blots shown in . Individual data points plotted ± SEM ( n = 3 WT, 4 SAKO mice per time point). * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

    Journal: Nature metabolism

    Article Title: Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3

    doi: 10.1038/s42255-020-0217-6

    Figure Lengend Snippet: a . Il6 expression in 12-week high fat diet (HFD) fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control for 30 minutes before sacrifice and tissue collection. Expression levels normalized to vehicle within each tissue. Individual data points plotted ± SEM ( n = 12 mice per treatment group). p value = 0.0003 (iWAT) and 0.006 (eWAT) b . Serum IL-6 levels in 12-week HFD fed C57BL/6 mice treated with 1 mg/kg CL-316,243 or vehicle control before sacrifice and blood collection. Data are represented as mean ± SEM ( n = 3 mice per genotype at each time point). p value < 0.0001 WT vs Il6 KO. Quantification of phosphorylation of c . HSL at serine 563 over total HSL ( p value < 0.0001 20 min vs. basal) d . p38 at threonine 180 and tyrosine 182 over total p38 ( p value = 0.003 20 min vs. basal) e . STAT3 at tyrosine 705 over total STAT3 ( p value < 0.0001 120 min vs. basal) and f . STAT3 at serine 727 over total STAT3 ( p value = 0.003 20 min vs. basal). c .- f . Western blots shown in . Individual data points plotted ± SEM ( n = 3 WT, 4 SAKO mice per time point). * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

    Article Snippet: STAT3 constructs in the pAdCMV vector were purchased from Addgene: pAdCMV/V5-DEST-STAT3–3xFlag (99260), pAdCMV/V5-DEST-S727A-STAT3–3xFlag (99262), pAdCMV/V5-DEST-S727D-STAT3–3xFlag (99263), and pAdCMV/V5-DEST-Y705F-STAT3–3xFlag (99261).

    Techniques: Expressing, Western Blot

    a ., f .- h . Western blot analysis of STAT3 phosphorylation in fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243; HSL phosphorylation in the lipid droplet fraction also shown to demonstrate effectiveness of CL-316,243 treatment. b . Western blot analysis of STAT3 phosphorylation in fractionated adipose tissue collected after intraperitoneal injection of 1 mg/kg CL-316,243 (20 or 120 min) or vehicle control (20 min after vehicle injection). c . Confocal images of PPDIVs stained with Bodipy, DAPI, STAT3 (Alex fluor 555 conjugated secondary) and pSer 727 STAT3 (Alex fluor 647 conjugated secondary). Individual channels of two representative vehicle and CL treated cells shown. Scale bar = 10 μm. d . 3-D representation of z-stack images of PPDIVs stained with bodipy (green), Dapi (blue) STAT3 (yellow) or pSer 727 STAT3 (red) are shown with colocalization of Bodipy and STAT3 shown in pink, and colocalization of Bodipy pSer 727 STAT3 shown in white. e . Quantification of colocalization of STAT3 and pSer 727 STAT3 with bodipy, normalized to lipid droplet volume (top) or lipid droplet surface area (bottom). Data are represented as mean ± SEM ( n = 61 V and 64 CL lipids droplets) p value < 0.0001 WT versus SAKO pSer 727 . f . Cells pretreated with H89 (PKA inhibitor) or JAK inhibitor I for 30 min, before Cl-316,243 treatment for 60 min. g . Cells pretreated with H89, SB-303,580 or atglistatin for 30 min, before Cl-316,243 treatment for 15 min. Right panel : Quantification of Ser 727 STAT3 phosphorylation in three independent experiments, data are represented as mean ± SEM. p value < 0.0001 DMSO v vs CL, CL DMSO vs. PKAi/ATGLi and = 0,003 p38i V vs CL. h . Cl-316,243 treatment performed in the presence or absence of 2% BSA in the media. Blots are representative of results from three independent experiments, 75 kDa and 100 kDa protein marker locations indicated in pink and blue respectively. Results in c - e were replicated in an independent experiment. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

    Journal: Nature metabolism

    Article Title: Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3

    doi: 10.1038/s42255-020-0217-6

    Figure Lengend Snippet: a ., f .- h . Western blot analysis of STAT3 phosphorylation in fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243; HSL phosphorylation in the lipid droplet fraction also shown to demonstrate effectiveness of CL-316,243 treatment. b . Western blot analysis of STAT3 phosphorylation in fractionated adipose tissue collected after intraperitoneal injection of 1 mg/kg CL-316,243 (20 or 120 min) or vehicle control (20 min after vehicle injection). c . Confocal images of PPDIVs stained with Bodipy, DAPI, STAT3 (Alex fluor 555 conjugated secondary) and pSer 727 STAT3 (Alex fluor 647 conjugated secondary). Individual channels of two representative vehicle and CL treated cells shown. Scale bar = 10 μm. d . 3-D representation of z-stack images of PPDIVs stained with bodipy (green), Dapi (blue) STAT3 (yellow) or pSer 727 STAT3 (red) are shown with colocalization of Bodipy and STAT3 shown in pink, and colocalization of Bodipy pSer 727 STAT3 shown in white. e . Quantification of colocalization of STAT3 and pSer 727 STAT3 with bodipy, normalized to lipid droplet volume (top) or lipid droplet surface area (bottom). Data are represented as mean ± SEM ( n = 61 V and 64 CL lipids droplets) p value < 0.0001 WT versus SAKO pSer 727 . f . Cells pretreated with H89 (PKA inhibitor) or JAK inhibitor I for 30 min, before Cl-316,243 treatment for 60 min. g . Cells pretreated with H89, SB-303,580 or atglistatin for 30 min, before Cl-316,243 treatment for 15 min. Right panel : Quantification of Ser 727 STAT3 phosphorylation in three independent experiments, data are represented as mean ± SEM. p value < 0.0001 DMSO v vs CL, CL DMSO vs. PKAi/ATGLi and = 0,003 p38i V vs CL. h . Cl-316,243 treatment performed in the presence or absence of 2% BSA in the media. Blots are representative of results from three independent experiments, 75 kDa and 100 kDa protein marker locations indicated in pink and blue respectively. Results in c - e were replicated in an independent experiment. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

    Article Snippet: STAT3 constructs in the pAdCMV vector were purchased from Addgene: pAdCMV/V5-DEST-STAT3–3xFlag (99260), pAdCMV/V5-DEST-S727A-STAT3–3xFlag (99262), pAdCMV/V5-DEST-S727D-STAT3–3xFlag (99263), and pAdCMV/V5-DEST-Y705F-STAT3–3xFlag (99261).

    Techniques: Western Blot, Injection, Staining, Marker

    a . Left panel: Western blot of mature adipocytes isolated from 12-week old WT and SAKO mice. Right panel: Quantification of STAT3 protein relative to RalA loading control. Individual data points plotted ± SEM ( n = 3 iWAT, 2 eWAT). b . Body weight of 12-week old ND fed WT and SAKO mice. Individual data points plotted ± SEM ( n = 6 per genotype). c . Oxygen consumption rate in ND fed WT and SAKO mice at 16-weeks of age. Data are represented as mean ± SEM ( n = 16). d . Adipocyte size distribution from ND-fed 12-week old WT and SAKO eWAT ( n = 2 WT and 3 SAKO). e . Adipocyte size distribution from ND-fed 12-week old WT and SAKO iWAT ( n = 2 WT and 3 SAKO). f . Body composition of ND fed WT and SAKO mice at 12 weeks of age. Individual data points plotted ± SEM ( n =6 WT and 4 SAKO).

    Journal: Nature metabolism

    Article Title: Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3

    doi: 10.1038/s42255-020-0217-6

    Figure Lengend Snippet: a . Left panel: Western blot of mature adipocytes isolated from 12-week old WT and SAKO mice. Right panel: Quantification of STAT3 protein relative to RalA loading control. Individual data points plotted ± SEM ( n = 3 iWAT, 2 eWAT). b . Body weight of 12-week old ND fed WT and SAKO mice. Individual data points plotted ± SEM ( n = 6 per genotype). c . Oxygen consumption rate in ND fed WT and SAKO mice at 16-weeks of age. Data are represented as mean ± SEM ( n = 16). d . Adipocyte size distribution from ND-fed 12-week old WT and SAKO eWAT ( n = 2 WT and 3 SAKO). e . Adipocyte size distribution from ND-fed 12-week old WT and SAKO iWAT ( n = 2 WT and 3 SAKO). f . Body composition of ND fed WT and SAKO mice at 12 weeks of age. Individual data points plotted ± SEM ( n =6 WT and 4 SAKO).

    Article Snippet: STAT3 constructs in the pAdCMV vector were purchased from Addgene: pAdCMV/V5-DEST-STAT3–3xFlag (99260), pAdCMV/V5-DEST-S727A-STAT3–3xFlag (99262), pAdCMV/V5-DEST-S727D-STAT3–3xFlag (99263), and pAdCMV/V5-DEST-Y705F-STAT3–3xFlag (99261).

    Techniques: Western Blot, Isolation

    a . Western blot analysis of fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243 or vehicle control for 60 min. b .- d . and f . Western blot analysis of input, flow through and immunoprecipitation using Myc-antibody coated beads ( b , c ) or Flag-antibody coated beads ( d , f ) of HEK293T cell lysates overexpressing Flag-tagged STAT3 and/or Myc-tagged GPAT3/GPAT4. Blots are representative of three independent replicates. Dark exposure (D.E.). e . Western blot analysis of input and immunoprecipitation using GPAT3 antibody in 3T3-L1 differentiated adipocytes treated with 10 μM CL-316,243 or vehicle control for 15 min. f . Western blot analysis of input, flow through and immunoprecipitation using Flag antibody coated beads of HEK293T cell lysates overexpressing Flag-tagged STAT3 (WT/S 727 A/S 727 D) and/or Myc-tagged GPAT3. Blots are representative of three independent replicates. Arrow indicates expected size of Ser 727 phosphorylated STAT3; the band observed in the IP samples is a larger non-specific band. g . Western blot analysis of input, flow through, and immunoprecipitation using Flag antibody coated beads from 3T3-L1 differentiated adipocytes with lentiviral overexpression of flag-tagged STAT3 (WT/Y 705 F/S 727 A) and/or Myc-tagged GPAT3, cells treated with 10 μM CL-316,243 or vehicle control for 60 min before harvest and IP. These experiments were repeated independently twice with similar results.

    Journal: Nature metabolism

    Article Title: Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3

    doi: 10.1038/s42255-020-0217-6

    Figure Lengend Snippet: a . Western blot analysis of fractionated 3T3-L1 adipocytes treated with 10 μM CL-316,243 or vehicle control for 60 min. b .- d . and f . Western blot analysis of input, flow through and immunoprecipitation using Myc-antibody coated beads ( b , c ) or Flag-antibody coated beads ( d , f ) of HEK293T cell lysates overexpressing Flag-tagged STAT3 and/or Myc-tagged GPAT3/GPAT4. Blots are representative of three independent replicates. Dark exposure (D.E.). e . Western blot analysis of input and immunoprecipitation using GPAT3 antibody in 3T3-L1 differentiated adipocytes treated with 10 μM CL-316,243 or vehicle control for 15 min. f . Western blot analysis of input, flow through and immunoprecipitation using Flag antibody coated beads of HEK293T cell lysates overexpressing Flag-tagged STAT3 (WT/S 727 A/S 727 D) and/or Myc-tagged GPAT3. Blots are representative of three independent replicates. Arrow indicates expected size of Ser 727 phosphorylated STAT3; the band observed in the IP samples is a larger non-specific band. g . Western blot analysis of input, flow through, and immunoprecipitation using Flag antibody coated beads from 3T3-L1 differentiated adipocytes with lentiviral overexpression of flag-tagged STAT3 (WT/Y 705 F/S 727 A) and/or Myc-tagged GPAT3, cells treated with 10 μM CL-316,243 or vehicle control for 60 min before harvest and IP. These experiments were repeated independently twice with similar results.

    Article Snippet: STAT3 constructs in the pAdCMV vector were purchased from Addgene: pAdCMV/V5-DEST-STAT3–3xFlag (99260), pAdCMV/V5-DEST-S727A-STAT3–3xFlag (99262), pAdCMV/V5-DEST-S727D-STAT3–3xFlag (99263), and pAdCMV/V5-DEST-Y705F-STAT3–3xFlag (99261).

    Techniques: Western Blot, Immunoprecipitation, Over Expression

    a . Incorporation of 14 C-palmitic acid into triglycerides by SAKO PPDIV relative to WT control cells ± 1 μM CL-316,243. Individual data points plotted ± SEM ( n = 3 wells per condition, p values = 0.0008). b . GPAT activity in iWAT homogenates from WT and SAKO mice treated with 1 mg/kg CL-316,243 or vehicle control for 20 minutes. Individual data points plotted ± SEM ( n = 3 mice per condition, p value =0.002 WT V vs. CL, 0.019 WT vs. SAKO CL). c . Western blot analysis of GPAT3 and GPAT4 protein levels in lysates from WT and SAKO iWAT and eWAT. Results are representative of three independent experiments, 50, 75 and 100 kDa protein markers indicated in green, pink and blue respectively. d . OCR at 30 min ± 10 μM CL-316,243 in 3T3-L1 adipocytes ± JAK inhibitor I pretreatment for 30 min, normalized to baseline. Individual data points plotted ± SEM ( n = 8 wells per condition, p values < 0.0001). e . and f . OCR at 30 min ± 0.5 μM CL-316,243 in WT and SAKO PPDIV with lentiviral STAT3 (WT, S 727 A or S 727 D) or GFP overexpression. e . Data are represented as mean ± SEM ( n = 12 wells per over expression construct in each genotype, p value < 0.0001 V vs. CL, 0.002 WT CL vs. SAKO CL GFP, and 0.046 WT CL vs. SAKO CL S727A ). f . Individual data points plotted ± SEM ( n = 4 wells per condition, p value = 0.029 V vs. CL STAT3, 0.016 V vs. CL 727D, 0.047 GFP vs. STAT3 CL, 0.011 727A vs. STAT3 CL, and 0.024 727A vs. 727D CL). g . Model of FA handling in the fed state (blue arrows) versus the fasted stated (red arrows) and the signaling regulating lipolysis-driven oxidative metabolism (black arrows) in the fasted state. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

    Journal: Nature metabolism

    Article Title: Catecholamines suppress fatty acid re-esterification and increase oxidation in white adipocytes via STAT3

    doi: 10.1038/s42255-020-0217-6

    Figure Lengend Snippet: a . Incorporation of 14 C-palmitic acid into triglycerides by SAKO PPDIV relative to WT control cells ± 1 μM CL-316,243. Individual data points plotted ± SEM ( n = 3 wells per condition, p values = 0.0008). b . GPAT activity in iWAT homogenates from WT and SAKO mice treated with 1 mg/kg CL-316,243 or vehicle control for 20 minutes. Individual data points plotted ± SEM ( n = 3 mice per condition, p value =0.002 WT V vs. CL, 0.019 WT vs. SAKO CL). c . Western blot analysis of GPAT3 and GPAT4 protein levels in lysates from WT and SAKO iWAT and eWAT. Results are representative of three independent experiments, 50, 75 and 100 kDa protein markers indicated in green, pink and blue respectively. d . OCR at 30 min ± 10 μM CL-316,243 in 3T3-L1 adipocytes ± JAK inhibitor I pretreatment for 30 min, normalized to baseline. Individual data points plotted ± SEM ( n = 8 wells per condition, p values < 0.0001). e . and f . OCR at 30 min ± 0.5 μM CL-316,243 in WT and SAKO PPDIV with lentiviral STAT3 (WT, S 727 A or S 727 D) or GFP overexpression. e . Data are represented as mean ± SEM ( n = 12 wells per over expression construct in each genotype, p value < 0.0001 V vs. CL, 0.002 WT CL vs. SAKO CL GFP, and 0.046 WT CL vs. SAKO CL S727A ). f . Individual data points plotted ± SEM ( n = 4 wells per condition, p value = 0.029 V vs. CL STAT3, 0.016 V vs. CL 727D, 0.047 GFP vs. STAT3 CL, 0.011 727A vs. STAT3 CL, and 0.024 727A vs. 727D CL). g . Model of FA handling in the fed state (blue arrows) versus the fasted stated (red arrows) and the signaling regulating lipolysis-driven oxidative metabolism (black arrows) in the fasted state. * p value < 0.05 from post hoc analysis after significant two-way ANOVA.

    Article Snippet: STAT3 constructs in the pAdCMV vector were purchased from Addgene: pAdCMV/V5-DEST-STAT3–3xFlag (99260), pAdCMV/V5-DEST-S727A-STAT3–3xFlag (99262), pAdCMV/V5-DEST-S727D-STAT3–3xFlag (99263), and pAdCMV/V5-DEST-Y705F-STAT3–3xFlag (99261).

    Techniques: Activity Assay, Western Blot, Over Expression, Construct