Prostaglandin synthesis and release by Doxo-induced senescent cells inhibits myoblast differentiation.

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, TNFα1, IL6, TGFβ1), and enzymes involved in the biosynthesis of prostaglandin PGD2/15d-PGJ2 (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. A representative widefield micrograph of cell morphology in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO.

F. Expression of mRNA of cell cycle inhibitor p21 and SASP factors (IL6 and TGFβ), measured by qPCR, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO.

G. Expression of cell cycle inhibitor p21, measured by immunoblot, in C2C12 myoblasts after 16 days of treatment with Doxo (150 nM) or DMSO.

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.

I. Expression of mRNAs of prostaglandin biosynthetic enzymes, measured by qPCR, in C2C12 myoblasts after treatment with Doxo (150 nM) or DMSO.

J. Concentration of 15d-PGJ2 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)

15d-PGJ2 inhibits differentiation of myoblasts.

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-PGJ2 (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-PGJ2 (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-PGJ2 (4 µM) or DMSO.

E. Expression of MHC protein, measured by immunoblotting, in primary human skeletal myoblasts after treatment with 15d-PGJ2 (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)

15d-PGJ2 covalently modifies HRas at Cysteine 184 and activates the HRas-MAPK pathway via the electrophilic cyclopentenone ring.

A. Streptavidin-immunoprecipitation of EGFP-HRas, measured by immunoblotting, in C2C12 cells after 3 hours of treatment with 15d-PGJ2-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-PGJ2 (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-PGJ2 (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-PGJ2 (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-PGJ2 (10 µM)/ 9,10-dihydro-15d-PGJ2 (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)

15d-PGJ2 controls the intracellular distribution of HRas and differentiation of C2C12 cells in an HRas C-terminal cysteine-dependent manner.

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 Rmean 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 Rmean, in C2C12 myoblasts treated with 15d-PGJ2 (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 Rmean, in C2C12 myoblasts treated with 15d-PGJ2 (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-PGJ2 (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-PGJ2 (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)

Treatment with Doxo induces senescence in vivo and in vitro and induces release of eicosanoid prostaglandin 15d-PGJ2

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. Representative peak of quantification of m/z 315.100 → m/z 271 transitions from blank samples.

D. Representative peak of quantification of m/z 315.100 → m/z 271 transitions from conditioned medium of C2C12 myoblasts treated with DMSO.

E. Representative peak of quantification of m/z 315.100 → m/z 271 transitions from conditioned medium of C2C12 myoblasts treated with Doxo (150 nM).

F. Standard curve of m/z 315.100 → m/z 271 fragment peak areas vs concentrations of 15d-PGJ2

Viability of C2C12 myoblasts after treatment with 15d-PGJ2 (10 µM) in differentiating medium.

A. Viability of C2C12 cells, measured by MTT assay, after 24 hours of treatment with 15-PGJ2 (0 µM, 1 µM, 2 µM, 4 µM, 5 µM, 10 µM).

(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)

Activation of HRas after treatment with 15d-PGJ2.

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-PGJ2 (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).

(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)

C-terminal cysteine-mediated intracellular distribution of constitutively active HRas (HRas V12) regulates the differentiation of C2C12 myoblasts.

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 Rmean, 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-PGJ2 or DMSO.

(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)

15d-PGJ2 conc. (pmol and fg/cell) detected in samples by mass spectrometry.

List of Primers:

List of Reagents

List of Antibodies