pgj 2  (Cayman Chemical)

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Schematic representation of the experimental flow for treatment of B6J mice with Doxo (5 mg/kg) or Saline. ( B ) Schematic representation of the experimental flow for treatment of senescent C2C12 cells with prostaglandin D synthase (PTGDS) inhibitor AT-56 (30 µM) or DMSO. ( C ) Morphology of MCF7 human breast adenocarcinoma cells after treatment with Doxo. ( D ) Expression and nuclear localization of cell cycle inhibitor p21 in MCF7 cells after 10 days of treatment with Doxo. ( E ) Nuclear area in MCF7 cells after 10 days of treatment with Doxo. ( F ) Expression of senescence-associated β-galactosidase activity (SA β-gal), measured by X-Gal staining in MCF7 cells after 10 days of treatment with Doxo. ( G ) Representative peak of quantification of m / z 315.100 ➔ m / z 271 transitions from blank samples. ( H ) Representative peak of quantification of m / z 315.100 ➔ m / z 271 transitions from conditioned medium of C2C12 myoblasts treated with DMSO. ( I ) Representative peak of quantification of m / z 315.100 ➔ m / z 271 transitions from conditioned medium of C2C12 myoblasts treated with Doxo (150 Nm). ( J ) Standard curve of m/z 315.100 ➔ m / z 271 fragment peak areas vs concentrations of 15d-PGJ 2 . (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ****p < 0.0001.)

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Saline, Expressing, Activity Assay, Staining, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Expression and localization of tumor suppressor protein p21, measured by immunofluorescence, in hindlimb skeletal muscles of mice after 11 days of treatment with Doxo (5 mg/kg) or Saline ( N = 3). ( B ) Representative confocal micrograph of expression of γH2A.X in the gastrocnemius muscle of mice treated with Doxo (5 mg/kg) or Saline ( N = 3). ( C ) Expression of mRNAs of senescence markers ( Cdkn2a and Cdkn1a ), senescence-associated secreted phenotype (SASP) factors ( Cxcl1 , Cxcl2 , Tnfa , Il6 , and Tgfb1 ), and enzymes involved in the biosynthesis of prostaglandin PGD 2 /15d-PGJ 2 ( Ptgs1 , Ptgs2 , and Ptgds ), measured by quantitative polymerase chain reaction (qPCR), in hindlimb skeletal muscles of mice after 11 days of treatment with Doxo (5 mg/kg) or Saline ( N = 4). ( D ) A representative confocal micrograph and a scatter plot of the nuclear area of C2C12 myoblasts, measured by immunofluorescence, after 16 days of treatment with Doxo (150 nM) or Dimethyl Sulphoxide (DMSO) ( N = 3). ( E ) A representative widefield micrograph of cell morphology in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( F ) Expression of mRNA of cell cycle inhibitor Cdkn1a and SASP factors ( Il6 and Tgfb1 ), measured by qPCR, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( G ) Expression of cell cycle inhibitor p21, measured by immunoblot, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( H ) Activity of senescence-associated β-galactosidase (SA β-gal), measured by X-gal staining at pH ~6, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( I ) Expression of mRNAs of prostaglandin biosynthetic enzymes, measured by qPCR, in C2C12 myoblasts after treatment with Doxo (150 nM) or DMSO ( N = 4). (Statistical significance was tested using Dunnett’s multiple comparisons test .) ( J ) Concentration of 15d-PGJ 2 released from quiescent or senescent C2C12 cells ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Figure 1—source data 1. Uncropped and labelled gels for . Figure 1—source data 2. Raw unedited gels for . Figure 1—source data 3. Dunnett’s multiple comparison test for the time-dependent expression of prostaglandin biosynthesis enzymes. C2C12 myoblasts treated with DMSO or Doxorubicin (Doxo) (150nM) from Day 0 to 16. Figure 1—source data 4. 15d-PGJ 2 concentration in the conditioned medium using mass spectrometry. Concentration of 15d-PGJ 2 (picograms (pg) and femtograms (fg)/cell) measured in the conditioned medium of quiescent and senescent C2C12 mouse myoblasts.

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Expressing, Immunofluorescence, Muscles, Saline, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Staining, Concentration Assay, Standard Deviation, Two Tailed Test, Comparison, Mass Spectrometry

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Expression of Myosin Heavy Chain (MHC) protein and the fusion of myoblasts in myotubes, measured by immunofluorescence, after treatment with conditioned medium of senescent cells treated with prostaglandin D synthase (PTGDS) inhibitor AT-56 (30 µM) or DMSO. ( B ) Normalized number of C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO ( N = 3). ( C ) Expression of mRNAs of markers of differentiation ( Myod1 , Myog , and Myhc ), measured by qPCR, in C2C12 myoblasts treated with 15d-PGJ 2 (1, 2, or 4 µM) or DMSO ( N = 3). ( D ) Expression of MHC protein and the fusion of myoblasts in syncytial myotubes, measured by immunofluorescence, after treatment with 15d-PGJ 2 (4 µM) or DMSO ( N = 3). ( E ) Expression of MHC protein, measured by immunoblotting, in primary human skeletal myoblasts after treatment with 15d-PGJ 2 (1, 2, or 4 µM) or DMSO for 5 days ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Figure 2—source data 1. Uncropped and labelled gels for . Figure 2—source data 2. Raw unedited gels for .

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Expressing, Immunofluorescence, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Streptavidin-immunoprecipitation of EGFP-HRas, measured by immunoblotting, in C2C12 cells after 3 hr of treatment with 15d-PGJ 2 -Biotin (5 µM) ( N = 3). ( B ) Representative confocal micrograph of fluorescence resonance energy transfer (FRET) between EGFP-tagged HRas (EGFP-HRas) and mCherry-tagged Ras-binding domain (RBD) of RAF kinase (mCherry-RAF-RBD). ( C ) Activation of the EGFP-tagged wild-type HRas (HRas WT), measured by FRET, before and after 1 hr of treatment with 15d-PGJ 2 (10 µM) after starvation for 24 hr. ( D ) Activation of the EGFP-tagged C-terminal cysteine mutants of HRas (HRas C181S and HRas C184S), measured by FRET, before and after 1 hr of treatment with 15d-PGJ 2 (10 µM) after starvation for 24 hr. ( E ) Phosphorylation of Erk (42 kDa and 44 kDa), measured by immunoblotting, in C2C12 cells after 1 hr of treatment with 15d-PGJ 2 (5 and 10 µM) or DMSO after starvation for 24 hr ( N = 3). ( F ) Phosphorylation of Erk (42 and 44 kDa), measured by immunoblotting, in C2C12 cells after 1 hr of treatment with 15d-PGJ 2 (10 µM)/ 9,10-dihydro-15d-PGJ 2 (10 µM) or DMSO after starvation for 24 hr ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001.) Figure 3—source data 1. Uncropped and labelled gels for . Figure 3—source data 2. Raw unedited gels for .

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Immunoprecipitation, Western Blot, Fluorescence, Förster Resonance Energy Transfer, Binding Assay, Activation Assay, Phospho-proteomics, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Representative confocal micrograph of C2C12 cells expressing only EGFP-tagged HRas or mCherry-tagged RAF-RBD alone for spectral overlap (bleed-through) calculations. ( B ) Mean bleed-through of EGFP-tagged HRas (Donor) and mCherry-tagged RAF-RBD (Acceptor) in the fluorescence resonance energy transfer (FRET) channel. ( C ) Phosphorylation of Akt (measured by immunoblotting) in C2C12 cells treated with 15d-PGJ 2 (5 and 10 µM) or DMSO for 1 hr after starving the cells in 0.2% serum medium for 24 hr. The densitometric ratio of phosphorylated Akt (Ser473) to total Akt was normalized to non-starved C2C12 cells. (The Standard Deviation between replicates was plotted as error bars. Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3; statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05.) Figure 3—figure supplement 1—source data 1. Uncropped and labelled gels for . Figure 3—figure supplement 1—source data 2. Raw unedited gels for .

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Expressing, Fluorescence, Förster Resonance Energy Transfer, Phospho-proteomics, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Representative confocal micrograph of C2C12 myoblasts showing localization of EGFP-tagged HRas between the plasma membrane (stained with Alexa Fluor 633-conjugated Wheat Germ Agglutinin) and the Golgi (labeled with TagRFP-tagged Golgi resident GalT protein). A statistic R mean was defined as the ratio of mean HRas intensity at the Golgi to the mean HRas intensity at the plasma membrane. ( B ) Distribution of the wild-type HRas between the Golgi and the plasma membrane, measured by R mean , in C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO for 24 hr. ( C ) Distribution of the C-terminal cysteine mutants of HRas between the Golgi and the plasma membrane, measured by R mean , in C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO for 24 hr. ( D ) Expression of mRNAs of known markers of differentiation ( Myod1 , Myog , and Myhc ), measured by qPCR, in differentiating C2C12 myoblasts expressing the EGFP-tagged wild-type and the C-terminal cysteine mutants of HRas after treatment with 15d-PGJ 2 (4 µM) or DMSO for 5 days ( N = 3). ( E ) Expression of MHC protein, measured by immunoblotting, in differentiating C2C12 myoblasts expressing the EGFP-tagged wild-type and the C-terminal cysteine mutants of HRas after treatment with 15d-PGJ 2 (4 µM) or DMSO for 5 days ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01.) Figure 4—source data 1. Uncropped and labelled gels for . Figure 4—source data 2. Raw unedited gels for .

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Clinical Proteomics, Membrane, Staining, Labeling, Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Distribution of EGFP-tagged HRas WT/HRas V12/HRas-C181S/HRas V12-C181S/HRas-C184S/HRas V12-C184S between the Golgi complex and the plasma membrane, as seen by the scatter plot of R mean , the ratio of the mean HRas intensity at the Golgi complex to that at the plasma membrane, in C2C12 cells. (Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3). ( B ) mRNA levels of Myod1 , Myog , and Myhc relative to 18s rRNA (measured by quantitative PCR) in C2C12 cells expressing EGFP-tagged HRas V12/HRas V12-C181S/HRas V12-C184S in C2C12 differentiation medium for 5 days. (Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3.) ( C ) Protein levels of MyHC (measured by immunoblotting) in C2C12 cells expressing EGFP-tagged HRas V12/HRas V12-C181S/HRas V12-C184S in C2C12 differentiation medium for 5 days. The densitometric ratio of levels of MyHC to α-Tubulin was normalized to C2C12 cells expressing HRas V12. (Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3.) ( D ) Brightfield image of C2C12 cells expressing EGFP-HRas V12, V12 C181S, or V12 C184S on Days 0 and 5 of differentiation. ( E ) Brightfield image of C2C12 cells expressing EGFP-HRas WT, C181S, or C184S after 5 days of treatment with 15d-PGJ 2 or DMSO. (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, ****p < 0.0001.) Figure 4—figure supplement 1—source data 1. Uncropped and labelled gels for . Figure 4—figure supplement 1—source data 2. Raw unedited gels for .

Article Snippet: 15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was diluted in DMEM media for experiments.

Techniques: Clinical Proteomics, Membrane, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Schematic representation of the experimental flow for treatment of B6J mice with Doxo (5 mg/kg) or Saline. ( B ) Schematic representation of the experimental flow for treatment of senescent C2C12 cells with prostaglandin D synthase (PTGDS) inhibitor AT-56 (30 µM) or DMSO. ( C ) Morphology of MCF7 human breast adenocarcinoma cells after treatment with Doxo. ( D ) Expression and nuclear localization of cell cycle inhibitor p21 in MCF7 cells after 10 days of treatment with Doxo. ( E ) Nuclear area in MCF7 cells after 10 days of treatment with Doxo. ( F ) Expression of senescence-associated β-galactosidase activity (SA β-gal), measured by X-Gal staining in MCF7 cells after 10 days of treatment with Doxo. ( G ) Representative peak of quantification of m / z 315.100 ➔ m / z 271 transitions from blank samples. ( H ) Representative peak of quantification of m / z 315.100 ➔ m / z 271 transitions from conditioned medium of C2C12 myoblasts treated with DMSO. ( I ) Representative peak of quantification of m / z 315.100 ➔ m / z 271 transitions from conditioned medium of C2C12 myoblasts treated with Doxo (150 Nm). ( J ) Standard curve of m/z 315.100 ➔ m / z 271 fragment peak areas vs concentrations of 15d-PGJ 2 . (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ****p < 0.0001.)

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Saline, Expressing, Activity Assay, Staining, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Expression and localization of tumor suppressor protein p21, measured by immunofluorescence, in hindlimb skeletal muscles of mice after 11 days of treatment with Doxo (5 mg/kg) or Saline ( N = 3). ( B ) Representative confocal micrograph of expression of γH2A.X in the gastrocnemius muscle of mice treated with Doxo (5 mg/kg) or Saline ( N = 3). ( C ) Expression of mRNAs of senescence markers ( Cdkn2a and Cdkn1a ), senescence-associated secreted phenotype (SASP) factors ( Cxcl1 , Cxcl2 , Tnfa , Il6 , and Tgfb1 ), and enzymes involved in the biosynthesis of prostaglandin PGD 2 /15d-PGJ 2 ( Ptgs1 , Ptgs2 , and Ptgds ), measured by quantitative polymerase chain reaction (qPCR), in hindlimb skeletal muscles of mice after 11 days of treatment with Doxo (5 mg/kg) or Saline ( N = 4). ( D ) A representative confocal micrograph and a scatter plot of the nuclear area of C2C12 myoblasts, measured by immunofluorescence, after 16 days of treatment with Doxo (150 nM) or Dimethyl Sulphoxide (DMSO) ( N = 3). ( E ) A representative widefield micrograph of cell morphology in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( F ) Expression of mRNA of cell cycle inhibitor Cdkn1a and SASP factors ( Il6 and Tgfb1 ), measured by qPCR, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( G ) Expression of cell cycle inhibitor p21, measured by immunoblot, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( H ) Activity of senescence-associated β-galactosidase (SA β-gal), measured by X-gal staining at pH ~6, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO ( N = 3). ( I ) Expression of mRNAs of prostaglandin biosynthetic enzymes, measured by qPCR, in C2C12 myoblasts after treatment with Doxo (150 nM) or DMSO ( N = 4). (Statistical significance was tested using Dunnett’s multiple comparisons test .) ( J ) Concentration of 15d-PGJ 2 released from quiescent or senescent C2C12 cells ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Figure 1—source data 1. Uncropped and labelled gels for . Figure 1—source data 2. Raw unedited gels for . Figure 1—source data 3. Dunnett’s multiple comparison test for the time-dependent expression of prostaglandin biosynthesis enzymes. C2C12 myoblasts treated with DMSO or Doxorubicin (Doxo) (150nM) from Day 0 to 16. Figure 1—source data 4. 15d-PGJ 2 concentration in the conditioned medium using mass spectrometry. Concentration of 15d-PGJ 2 (picograms (pg) and femtograms (fg)/cell) measured in the conditioned medium of quiescent and senescent C2C12 mouse myoblasts.

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Expressing, Immunofluorescence, Muscles, Saline, Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Staining, Concentration Assay, Standard Deviation, Two Tailed Test, Comparison, Mass Spectrometry

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Expression of Myosin Heavy Chain (MHC) protein and the fusion of myoblasts in myotubes, measured by immunofluorescence, after treatment with conditioned medium of senescent cells treated with prostaglandin D synthase (PTGDS) inhibitor AT-56 (30 µM) or DMSO. ( B ) Normalized number of C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO ( N = 3). ( C ) Expression of mRNAs of markers of differentiation ( Myod1 , Myog , and Myhc ), measured by qPCR, in C2C12 myoblasts treated with 15d-PGJ 2 (1, 2, or 4 µM) or DMSO ( N = 3). ( D ) Expression of MHC protein and the fusion of myoblasts in syncytial myotubes, measured by immunofluorescence, after treatment with 15d-PGJ 2 (4 µM) or DMSO ( N = 3). ( E ) Expression of MHC protein, measured by immunoblotting, in primary human skeletal myoblasts after treatment with 15d-PGJ 2 (1, 2, or 4 µM) or DMSO for 5 days ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Figure 2—source data 1. Uncropped and labelled gels for . Figure 2—source data 2. Raw unedited gels for .

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Expressing, Immunofluorescence, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Streptavidin-immunoprecipitation of EGFP-HRas, measured by immunoblotting, in C2C12 cells after 3 hr of treatment with 15d-PGJ 2 -Biotin (5 µM) ( N = 3). ( B ) Representative confocal micrograph of fluorescence resonance energy transfer (FRET) between EGFP-tagged HRas (EGFP-HRas) and mCherry-tagged Ras-binding domain (RBD) of RAF kinase (mCherry-RAF-RBD). ( C ) Activation of the EGFP-tagged wild-type HRas (HRas WT), measured by FRET, before and after 1 hr of treatment with 15d-PGJ 2 (10 µM) after starvation for 24 hr. ( D ) Activation of the EGFP-tagged C-terminal cysteine mutants of HRas (HRas C181S and HRas C184S), measured by FRET, before and after 1 hr of treatment with 15d-PGJ 2 (10 µM) after starvation for 24 hr. ( E ) Phosphorylation of Erk (42 kDa and 44 kDa), measured by immunoblotting, in C2C12 cells after 1 hr of treatment with 15d-PGJ 2 (5 and 10 µM) or DMSO after starvation for 24 hr ( N = 3). ( F ) Phosphorylation of Erk (42 and 44 kDa), measured by immunoblotting, in C2C12 cells after 1 hr of treatment with 15d-PGJ 2 (10 µM)/ 9,10-dihydro-15d-PGJ 2 (10 µM) or DMSO after starvation for 24 hr ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001.) Figure 3—source data 1. Uncropped and labelled gels for . Figure 3—source data 2. Raw unedited gels for .

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Immunoprecipitation, Western Blot, Fluorescence, Förster Resonance Energy Transfer, Binding Assay, Activation Assay, Phospho-proteomics, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Representative confocal micrograph of C2C12 cells expressing only EGFP-tagged HRas or mCherry-tagged RAF-RBD alone for spectral overlap (bleed-through) calculations. ( B ) Mean bleed-through of EGFP-tagged HRas (Donor) and mCherry-tagged RAF-RBD (Acceptor) in the fluorescence resonance energy transfer (FRET) channel. ( C ) Phosphorylation of Akt (measured by immunoblotting) in C2C12 cells treated with 15d-PGJ 2 (5 and 10 µM) or DMSO for 1 hr after starving the cells in 0.2% serum medium for 24 hr. The densitometric ratio of phosphorylated Akt (Ser473) to total Akt was normalized to non-starved C2C12 cells. (The Standard Deviation between replicates was plotted as error bars. Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3; statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05.) Figure 3—figure supplement 1—source data 1. Uncropped and labelled gels for . Figure 3—figure supplement 1—source data 2. Raw unedited gels for .

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Expressing, Fluorescence, Förster Resonance Energy Transfer, Phospho-proteomics, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Representative confocal micrograph of C2C12 myoblasts showing localization of EGFP-tagged HRas between the plasma membrane (stained with Alexa Fluor 633-conjugated Wheat Germ Agglutinin) and the Golgi (labeled with TagRFP-tagged Golgi resident GalT protein). A statistic R mean was defined as the ratio of mean HRas intensity at the Golgi to the mean HRas intensity at the plasma membrane. ( B ) Distribution of the wild-type HRas between the Golgi and the plasma membrane, measured by R mean , in C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO for 24 hr. ( C ) Distribution of the C-terminal cysteine mutants of HRas between the Golgi and the plasma membrane, measured by R mean , in C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO for 24 hr. ( D ) Expression of mRNAs of known markers of differentiation ( Myod1 , Myog , and Myhc ), measured by qPCR, in differentiating C2C12 myoblasts expressing the EGFP-tagged wild-type and the C-terminal cysteine mutants of HRas after treatment with 15d-PGJ 2 (4 µM) or DMSO for 5 days ( N = 3). ( E ) Expression of MHC protein, measured by immunoblotting, in differentiating C2C12 myoblasts expressing the EGFP-tagged wild-type and the C-terminal cysteine mutants of HRas after treatment with 15d-PGJ 2 (4 µM) or DMSO for 5 days ( N = 3). (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, **p < 0.01.) Figure 4—source data 1. Uncropped and labelled gels for . Figure 4—source data 2. Raw unedited gels for .

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Clinical Proteomics, Membrane, Staining, Labeling, Expressing, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Senescent cells inhibit mouse myoblast differentiation via the SASP-lipid 15d-PGJ 2 mediated modification and control of HRas

doi: 10.7554/eLife.95229

Figure Lengend Snippet: ( A ) Distribution of EGFP-tagged HRas WT/HRas V12/HRas-C181S/HRas V12-C181S/HRas-C184S/HRas V12-C184S between the Golgi complex and the plasma membrane, as seen by the scatter plot of R mean , the ratio of the mean HRas intensity at the Golgi complex to that at the plasma membrane, in C2C12 cells. (Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3). ( B ) mRNA levels of Myod1 , Myog , and Myhc relative to 18s rRNA (measured by quantitative PCR) in C2C12 cells expressing EGFP-tagged HRas V12/HRas V12-C181S/HRas V12-C184S in C2C12 differentiation medium for 5 days. (Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3.) ( C ) Protein levels of MyHC (measured by immunoblotting) in C2C12 cells expressing EGFP-tagged HRas V12/HRas V12-C181S/HRas V12-C184S in C2C12 differentiation medium for 5 days. The densitometric ratio of levels of MyHC to α-Tubulin was normalized to C2C12 cells expressing HRas V12. (Statistical significance tested by two-tailed heteroscedastic Student’s t -test, N = 3.) ( D ) Brightfield image of C2C12 cells expressing EGFP-HRas V12, V12 C181S, or V12 C184S on Days 0 and 5 of differentiation. ( E ) Brightfield image of C2C12 cells expressing EGFP-HRas WT, C181S, or C184S after 5 days of treatment with 15d-PGJ 2 or DMSO. (The Standard Deviation between replicates was plotted as error bars. Statistical significance was tested by the two-tailed Student’s t -test ns = p > 0.05, *p < 0.05, ****p < 0.0001.) Figure 4—figure supplement 1—source data 1. Uncropped and labelled gels for . Figure 4—figure supplement 1—source data 2. Raw unedited gels for .

Article Snippet: 9,10-dihydro-15d-PGJ 2 (Cayman Chemical Company) dissolved in DMSO (10 mM) was appropriately diluted in DMEM media for experiments.

Techniques: Clinical Proteomics, Membrane, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

Journal: PNAS Nexus

Article Title: A new type of sulfation reaction: C -sulfonation for α,β-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1

doi: 10.1093/pnasnexus/pgae097

Figure Lengend Snippet: Kinetic constants of the sulfonation mediated by the mouse SULT7A1. a

Article Snippet: To prepare sulfonated 2-cyclopentenone (Sigma-Aldrich, Tokyo, Japan) and 15d-PGJ 2 (Cayman Chemical, Ann Arbor, MI, USA) for MS and NMR analyses, the above-mentioned sulfotransferase reaction was performed at larger scale with nonradioactive PAPS and an extended reaction time (3 h).

Techniques:

Journal: PNAS Nexus

Article Title: A new type of sulfation reaction: C -sulfonation for α,β-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1

doi: 10.1093/pnasnexus/pgae097

Figure Lengend Snippet: Sulfonation of cyclopentenone prostaglandins as mediated by SULT7A1. a) Specific sulfonating activity of SULT7A1 toward cyclopentenone prostaglandins (100 μM substrate concentration). Data represent the mean ± SD derived from three determinations. b) Analysis of [ 35 S]sulfonated products of prostaglandins generated by SULT7A1-expressing BHK cells. The figure shows the autoradiograph taken from the TLC plate used for the analysis of spent labeling media. Lanes 1 and 2 correspond to the labeling media of cells transfected with pEF6 (lane 1) or pEF6-SULT7A1 (lane 2). CT (Control) refers to the control-labeling medium without added prostaglandins. The arrows indicate the sulfonated derivatives. c) MS/MS analysis of the sulfonated product of 15d-PGJ 2 . The MS/MS spectrum is obtained from a parent ion peak at 397.17 m / z . d) 1 H-NMR analysis of the sulfated product of 15d-PGJ 2 . Each proton numbered in chemical structures is assigned for the corresponding spectrum.

Article Snippet: To prepare sulfonated 2-cyclopentenone (Sigma-Aldrich, Tokyo, Japan) and 15d-PGJ 2 (Cayman Chemical, Ann Arbor, MI, USA) for MS and NMR analyses, the above-mentioned sulfotransferase reaction was performed at larger scale with nonradioactive PAPS and an extended reaction time (3 h).

Techniques: Activity Assay, Concentration Assay, Derivative Assay, Generated, Expressing, Autoradiography, Labeling, Transfection, Control, Tandem Mass Spectroscopy

Journal: PNAS Nexus

Article Title: A new type of sulfation reaction: C -sulfonation for α,β-unsaturated carbonyl groups by a novel sulfotransferase SULT7A1

doi: 10.1093/pnasnexus/pgae097

Figure Lengend Snippet: The effect of the sulfonation on the ligand activity of the 15d-PGJ 2 toward prostanoid receptors. a) Agonist activity of sulfonated 15d-PGJ 2 toward DP1, EP2, EP4, and IP. cAMP formed in cells in the presence of the corresponding ligand prostaglandins (10 nM), 15d-PGJ 2 (100 or 1,000 nM), or sulfonated 15d-PGJ 2 (100 or 1,000 nM) is expressed as relative values (%) against the amount of cAMP formed in the presence of the corresponding ligand prostaglandins. b) Antagonist activity of sulfonated 15d-PGJ 2 toward DP1, EP2, EP4, and IP. cAMP formed in 293 T cells in the presence of the corresponding ligand prostaglandins (PGD 2 : 0.1 nM, PGE 2 : 0.05 nM, or PGI 2 : 10 nM) plus 15d-PGJ 2 (100 or 1,000 nM), or sulfonated 15d-PGJ 2 (100 or 1,000 nM) is expressed as relative values (%) against the amount of cAMP formed in the presence of the corresponding ligand prostaglandins. Statistical significance vs. the control sample (the corresponding ligand prostaglandins in the absence of 15d-PGJ 2 and sulfonate 15d-PGJ 2 ) is indicated by * P < 0.05 as analyzed by one-way ANOVA with Dunnett’s test. ND refers to the activity not detected.

Article Snippet: To prepare sulfonated 2-cyclopentenone (Sigma-Aldrich, Tokyo, Japan) and 15d-PGJ 2 (Cayman Chemical, Ann Arbor, MI, USA) for MS and NMR analyses, the above-mentioned sulfotransferase reaction was performed at larger scale with nonradioactive PAPS and an extended reaction time (3 h).

Techniques: Activity Assay, Control

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Schematic Representation of the experimental flow for treatment of B6J mice with Doxo (5 mg/kg) or Saline. B. Schematic representation of the experimental flow for treatment of senescent C2C12 cells with prostaglandin D synthase (PTGDS) inhibitor AT-56 (30 µM) or DMSO. C. Widefield micrograph of the morphology of C2C12 cells after treatment with Doxo (150 nM) or DMSO. D. Widefield micrograph of expression of Senescence Associated β-galactosidase (SA β-gal), measured by X-gal staining, in C2C12 cells after 13 days of treatment with Doxo (150 nM) or DMSO. E. Representative peaks from Blank sample. F. Representative peaks from Conditioned medium of C2C12 myoblasts treated with DMSO. G. Representative peaks from Conditioned medium of C2C12 myoblasts treated with Doxo (150 nM). H. Standard curve of peak area vs known conc. of 15d-PGJ 2 detected using AB SCIEX QTRAP 6500 mass spectrometer.

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Saline, Expressing, Staining, Mass Spectrometry

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Expression and localization of tumor suppressor protein p21, measured by immunofluorescence, in hindlimb skeletal muscles of mice after 11 days of treatment with Doxo (5 mg/kg) or Saline. B. Representative confocal micrograph of expression of γH2A.X in the gastrocnemius muscle of mice treated with Doxo (5 mg/kg) or Saline. C. Expression of mRNAs of senescence markers (p16 and p21), SASP factors (CXCL1, CXCL2, TNF1α, IL6, TGFβ1), and enzymes involved in the biosynthesis of prostaglandin PGD 2 /15d-PGJ 2 (PTGS1, PTGS2, PTGDS), measured by qPCR, in hindlimb skeletal muscles of mice after 11 days of treatment with Doxo (5 mg/kg) or Saline. D. A representative confocal micrograph and a scatter plot of the nuclear area of C2C12 myoblasts, measured by immunofluorescence, after 16 days of treatment with Doxo (150 nM) or DMSO. E. Expression of mRNAs of prostaglandin biosynthetic enzymes, measured by qPCR, in C2C12 myoblasts after treatment with Doxo (150 nM) or DMSO. F. Concentration of 15d-PGJ 2 released from quiescent or senescent C2C12 cells. (Statistical significance was tested by the two-tailed student’s t-test ns=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001)

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Expressing, Immunofluorescence, Muscles, Saline, Concentration Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Expression of MHC protein and the fusion of myoblasts in myotubes, measured by immunofluorescence, after treatment with conditioned medium of senescent cells treated with PTGDS inhibitor AT-56 (30 µM) or DMSO B. Normalized number of C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO C. Expression of mRNAs of markers of differentiation (MyoD, MyoG, and MHC), measured by qPCR, in C2C12 myoblasts treated with 15d-PGJ 2 (1 µM, 2 µM, or 4 µM) or DMSO. D. Expression of MHC protein and the fusion of myoblasts in syncytial myotubes, measured by immunofluorescence, after treatment with 15d-PGJ 2 (4 µM) or DMSO. E. Expression of MHC protein, measured by immunoblotting, in primary human skeletal myoblasts after treatment with 15d-PGJ 2 (1 µM, 2 µM, or 4 µM) or DMSO for 5 days. (Statistical significance was tested by the two-tailed student’s t-test ns=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001)

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Expressing, Immunofluorescence, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Representative images taken every 24 hours of C2C12 treated with 15d-PGJ 2 (5 and 10 µM) or DMSO in the C2C12 differentiation medium. B. Representative images taken every 24 hours of C2C12 treated with 15d-PGJ 2 (1, 2, and 4 µM) or DMSO in the C2C12 differentiation medium.

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques:

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Streptavidin-immunoprecipitation of EGFP-HRas, measured by immunoblotting, in C2C12 cells after 3 hours of treatment with 15d-PGJ 2 -Biotin (5 µM). B. Representative confocal micrograph of Fluorescence Resonance Energy Transfer (FRET) between EGFP-tagged HRas (EGFP-HRas) and mCherry-tagged Ras binding domain (RBD) of RAF kinase (mCherry-RAF RBD). C. Activation of the EGFP-tagged wild type HRas (HRas WT), measured by FRET, before and after 1 hour of treatment with 15d-PGJ 2 (10 µM) after starvation for 24 hours. D. Activation of the EGFP-tagged C-terminal cysteine mutants of HRas (HRas C181S and HRas C184S), measured by FRET, before and after 1 hour of treatment with 15d-PGJ 2 (10 µM) after starvation for 24 hours. E. Phosphorylation of Erk (42 kDa and 44 kDa), measured by immunoblotting, in C2C12 cells after 1 hour of treatment with 15d-PGJ 2 (5 µM, 10 µM) or DMSO after starvation for 24 hours. F. Phosphorylation of Erk (42 kDa and 44 kDa), measured by immunoblotting, in C2C12 cells after 1 hour of treatment with 15d-PGJ 2 (10 µM)/ 9,10-dihydro-15d-PGJ 2 (10 µM) or DMSO after starvation for 24 hours. (Statistical significance was tested by the two-tailed student’s t-test ns=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001)

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Immunoprecipitation, Western Blot, Fluorescence, Förster Resonance Energy Transfer, Binding Assay, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Representative confocal micrograph of C2C12 cells expressing only EGFP-tagged HRas or mCherry-tagged RAF RBD alone for spectral overlap (bleed-through) calculations. B. Mean bleed-through of EGFP-tagged HRas (Donor) and mCherry-tagged RAF RBD (Acceptor) in the FRET channel. C. Phosphorylation of Akt (measured by immunoblotting) in C2C12 cells treated with 15d-PGJ 2 (5, 10 µM) or DMSO for 1 hr after starving the cells in 0.2% serum medium for 24 hrs. The densitometric ratio of phosphorylated Akt (Ser473) to total Akt was normalized to non-starved C2C12 cells. (Statistical significance tested by two-tailed heteroscedastic student’s t-test, N=3).

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Representative confocal micrograph of C2C12 myoblasts showing localization of EGFP-tagged HRas between the plasma membrane (stained with Alexa Fluor 633 conjugated Wheat Germ Agglutinin) and the Golgi (labelled with TagRFP-tagged Golgi resident GalT protein). A statistic R mean was defined as the ratio of mean HRas intensity at the Golgi to the mean HRas intensity at the plasma membrane. B. Distribution of the wild-type HRas between the Golgi and the plasma membrane, measured by R mean , in C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO for 24 hours. C. Distribution of the C-terminal cysteine mutants of HRas between the Golgi and the plasma membrane, measured by R mean , in C2C12 myoblasts treated with 15d-PGJ 2 (10 µM) or DMSO for 24 hours. D. Expression of mRNAs of known markers of differentiation (MyoD, MyoG, and MHC), measured by qPCR, in differentiating C2C12 myoblasts expressing the EGFP-tagged wild-type and the C-terminal cysteine mutants of HRas after treatment with 15d-PGJ 2 (4 µM) or DMSO for 5 days. E. Expression of MHC protein, measured by immunoblotting, in differentiating C2C12 myoblasts expressing the EGFP-tagged wild-type and the C-terminal cysteine mutants of HRas after treatment with 15d-PGJ 2 (4 µM) or DMSO for 5 days. (Statistical significance was tested by the two-tailed student’s t-test ns=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001)

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Membrane, Staining, Expressing, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet: A. Distribution of EGFP-tagged HRas WT/ HRas V12/ HRas-C181S/ HRas V12-C181S/ HRas-C184S/ HRas V12-C184S between the Golgi complex and the plasma membrane, as seen by the scatter plot of R mean , the ratio of the mean HRas intensity at the Golgi complex to that at the plasma membrane, in C2C12 cells. (Statistical significance tested by two-tailed heteroscedastic student’s t-test, N=3) B. mRNA levels of MyoD, MyoG, and MyHC relative to 18s rRNA (measured by quantitative PCR) in C2C12 cells expressing EGFP-tagged HRas V12/ HRas V12-C181S/ HRas V12-C184S in C2C12 differentiation medium for 5 days. (Statistical significance tested by two-tailed heteroscedastic student’s t-test, N=3) C. Protein levels of MyHC (measured by immunoblotting) in C2C12 cells expressing EGFP-tagged HRas V12/HRas V12-C181S/HRas V12-C184S in C2C12 differentiation medium for 5 days. The densitometric ratio of levels of MyHC to α-Tubulin was normalized to C2C12 cells expressing HRas V12. (Statistical significance tested by two-tailed heteroscedastic student’s t-test, N=3). D. Brightfield image of C2C12 cells expressing EGFP-HRas V12, V12 C181S, or V12 C184S on Day 0 and Day 5 of differentiation. E. Brightfield image of C2C12 cells expressing EGFP-HRas WT, C181S, or C184S after 5 days of treatment with 15d-PGJ 2 or DMSO.

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Membrane, Two Tailed Test, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: bioRxiv

Article Title: Senescent cells inhibit muscle differentiation via the lipid- SASP 15d-PGJ 2 mediated modification and control of HRas

doi: 10.1101/2023.12.23.573200

Figure Lengend Snippet:

Article Snippet: C2C12 cells transfected with EGFP-HRas WT/C181S/C184S in 35mm dishes were treated with 15d-PGJ 2 -Biotin in DMEM Hi Glucose medium (Gibco) supplemented with 1% Penicilin-streptomycin-Glutamine (Gibco) without fetal bovine serum for 3 hours.

Techniques: Mass Spectrometry

Journal: bioRxiv

Article Title: Host-derived oxidized phospholipids initiate effector-triggered immunity fostering lethality upon microbial encounter

doi: 10.1101/2023.11.21.568047

Figure Lengend Snippet: A ) Schematic shows upstream and downstream kinases in the AKT signaling network. B ) BMDMs were primed, or not, with LPS and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). Phosphorylation of upstream AKT regulators (TBK1, IKKε, PDK1, PTEN and PI3K) was analyzed by immunoblot. Images are representative of three independent experiments. C ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and then stimulated with oxPAPC (100, 50 or 25 μg ml −1 ). The phosphorylation of the indicated mTORC1/2-associated proteins was analyzed after 1h by immunoblotting. Data are representative of three independent experiments. D ) WT and Nfe2l2 −/− BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with oxPAPC (100 μg ml −1 ) for 1h. The indicated transcripts were analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. Statistical significance was calculated using two-way ANOVA and Sidak’s multiple comparisons test. E-F ) BMDMs were primed, or not, with LPS (1 μg ml −1 ) and treated or not with 15d-PGJ 2 (20, 10 or 1 μM in E and 10 μM in F ) for 1h ( E ) or 24h ( F ). AKT phosphorylation and NRF2 accumulation were assessed by immunoblot ( E ). Data are representative of three independent experiments. IL-10 and TNF release was quantified by ELISA ( F ). n = 6, graphs are representative of three independent experiments and show means ± SEM. Statistical multiple comparisons were calculated by two-way ANOVA and Sidak’s test. G ) Expression of Akt isoforms (RNA-seq, Immgen.org database) in the indicated murine macrophage populations. H ) Normalized expression of Akt1 , Akt2 and Akt3 in BMDMs, analyzed by qPCR. n = 3, data are representative of three independent experiments and show means ± SEM. I, J ) Microscale thermophoresis (MST) analysis of oxPAPC interactions with AKT2 ( I ) and AKT3 ( J ). Traces of fluorescently labeled human recombinant AKT incubated with oxPAPC (1000, 500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM) or DPPC (500, 250,125, 62.5, 31.25, 15.62, 7.8, 3.9, 1.9, 0.97, 0.488, 0.24, 0.12, 0.0061 and 0.030 μM). oxPAPC-AKT binding curve was derived from the quantification of normalized fluorescence changes. n = 3, graph shows means ± SD. Images are representative of three independent experiments. K, L ) Active human recombinant AKT2 ( K ) or AKT3 ( L ) were incubated with oxPAPC and a kinase-specific FRET-peptide substrate (Z-’LYTE). Kinase inhibition was measured as the ability of lipid to block substrate phosphorylation. n = 4, graph shows means ± SD. Data are representative of three independent experiments.

Article Snippet: 15d-PGJ 2 (Cat# 18570) and 2-deoxy-D-Glucose (2-DG, Cat# 14325) were purchased from Cayman Chemicals.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, RNA Sequencing Assay, Microscale Thermophoresis, Labeling, Recombinant, Incubation, Binding Assay, Derivative Assay, Fluorescence, Inhibition, Blocking Assay