Journal: Archives of toxicology

Article Title: Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity

doi: 10.1007/s00204-019-02405-w

Figure Lengend Snippet: (A and B) Cultured neonatal cardiomyocytes were infected with Ad-MKP1 or Ad-gal in combination with Ad-Pten. After 24 hours of infection, cardiomyocytes were incubated with doxorubicin (1 μmol/L) or saline for 24 hours. Caspase-3 activity (a) and DNA fragmentation (b) were measured. (c and d) Cultured neonatal cardiomyocytes were transfected with MKP-1 siRNA or a scrambled siRNA as a control (NC siRNA). Cardiomyocytes were then infected with Ad-Capn2 or Ad-gal for 24 hours, followed by incubation with doxorubicin (1 μmol/L) for an additional 24 hours. Caspase-3 activity (c) and DNA fragmentation (d) were measured. Data are mean ± SD, n=5-6 independent cell cultures. *P < 0.05 versus Ad-gal or Ad-gal + NC siRNA, †P < 0.05 versus Doxorubicin + Ad-gal or Doxorubicin +Ad-gal + NC siRNA + Doxorubicin, and ‡P < 0.05 versus Doxorubicin + Ad-Pten or Doxorubicin+Ad-Capn2 + NC siRNA.

Article Snippet: Cultured neonatal cardiomyocytes were infected with adenoviral vectors containing capn1, capn2, pten and mkp-1 gene (Ad-Capn2, Ad-Capn1, Ad-Pten, Ad-MKP-1, SignaGen, USA) or β-gal (Ad-gal, Vector Biolabs, USA) as a control at a multiplicity of infection of 100 plaque forming units/cell ( Peng et al. 2003 ).

Techniques: Cell Culture, Infection, Incubation, Saline, Activity Assay, Transfection, Control

Journal: Archives of toxicology

Article Title: Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity

doi: 10.1007/s00204-019-02405-w

Figure Lengend Snippet: Cultured neonatal cardiomyocytes were infected with Ad-Capn2 or Ad-gal. (a) A representative western blot for Akt and phosphorylated Akt (Ser308 and Thr473) from 3 different cell cultures. (b) Cultured neonatal cardiomyocytes were infected with Ad-Capn2 or Ad-gal in the presence of LY294002 (LY, 2μmol/L) or Vehicle for the indicated times. Upper panel; A representative western blot from 4 different cell cultures for MKP-1 and GAPDH protein, lower panel; quantitation of MKP-1/GAPDH ratio. (c) Twenty-four hours after Ad-Capn2 infection, the cells were incubated with cycloheximide (CHX, 5 μg/mL) for the indicated times. Upper panel; A representative western blot from 4 independent cell cultures for MKP-1 and GAPDH protein, lower panel; quantitation of MKP-1/GAPDH ratio. Data are mean ± SD, n=4 independent cell cultures. *P < 0.05 versus Ad-gal+Vehicle or Ad-Capn2+LY and †P < 0.05 versus Ad-Capn2+Vehicle. (d) Cultured neonatal cardiomyocytes were infected with Ad-Capn2 or Ad-gal. Upper panel; A representative western blot for PTEN and GAPDH from 4 independent cell cultures, lower panel; quantitation of PTEN/GAPDH ratio. (e) Cardiomyocytes were infected with Ad-Pten or Ad-gal, followed by Ad-Capn2. Upper panel; A representative western blot for phosphorylated Akt and total Akt, lower panels; quantitation of phosphorylated Akt/Akt ratio. Data are mean ± SD, n=4 independent cell cultures. *P < 0.05 versus Ad-gal and †P < 0.05 versus Ad-Capn2+Ad-gal.

Article Snippet: Cultured neonatal cardiomyocytes were infected with adenoviral vectors containing capn1, capn2, pten and mkp-1 gene (Ad-Capn2, Ad-Capn1, Ad-Pten, Ad-MKP-1, SignaGen, USA) or β-gal (Ad-gal, Vector Biolabs, USA) as a control at a multiplicity of infection of 100 plaque forming units/cell ( Peng et al. 2003 ).

Techniques: Cell Culture, Infection, Western Blot, Quantitation Assay, Incubation

Journal: Archives of toxicology

Article Title: Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity

doi: 10.1007/s00204-019-02405-w

Figure Lengend Snippet: (a and b) Cultured neonatal cardiomyocytes were infected with Ad-Capn2 or Ad-gal in combination with Ad-Pten. After 24 hours of infection, cardiomyocytes were incubated with doxorubicin (1μmol/L) or saline for 24 hours. Caspase-3 activity (a) and DNA fragmentation (b) were measured. (c and d) Cultured neonatal cardiomyocytes were infected with Ad-Capn2 or Ad-gal in combination with LY294002 (2 μmol/L) or Vehicle for 24 hours, and then incubated with doxorubicin (1 μmol/L) for additional 24 hours. Caspase-3 activity (c) and DNA fragmentation (d) were measured. Data are mean ± SD, n=4-6 independent cell cultures. *P < 0.05 versus Ad-gal or Ad-gal + Vehicle, †P < 0.05 versus Ad-gal + Doxorubicin and ‡P < 0.05 versus Ad-Capn2 + Doxorubicin.

Article Snippet: Cultured neonatal cardiomyocytes were infected with adenoviral vectors containing capn1, capn2, pten and mkp-1 gene (Ad-Capn2, Ad-Capn1, Ad-Pten, Ad-MKP-1, SignaGen, USA) or β-gal (Ad-gal, Vector Biolabs, USA) as a control at a multiplicity of infection of 100 plaque forming units/cell ( Peng et al. 2003 ).

Techniques: Cell Culture, Infection, Incubation, Saline, Activity Assay

Journal: Archives of toxicology

Article Title: Calpain-2 promotes MKP-1 expression protecting cardiomyocytes in both in vitro and in vivo mouse models of doxorubicin-induced cardiotoxicity

doi: 10.1007/s00204-019-02405-w

Figure Lengend Snippet: Tg-Capn2/tTA (TG) and Tg-Capn2 mice (WT) received doxorubicin or saline. Five days later, heart tissues were analyzed for PTEN, Akt and MKP-1 protein expression. (a) Upper panel; representative western blot for PTEN and GAPDH from 2 out of 5 different hearts from WT and TG at 5 days, lower panel: quantitation of MKP-1/GAPDH ratio. (b) Upper panel; representative western blot for Akt and phosphorylated Akt (Ser308 and Thr473) from 2 out of 6 different hearts form WT and TG, lower panel; quantitation of phosphorylated Akt/Akt ratio. (c) Upper panel; representative western blot for MKP-1 and GAPDH from 2 out of 5 different hearts from WT and TG, lower panel: quantitation of MKP-1/GAPDH ratio. Data are mean ± SD, n=5-6 different hearts. *P < 0.05 versus WT + saline and †P < 0.05 versus WT + Doxorubicin.

Article Snippet: Cultured neonatal cardiomyocytes were infected with adenoviral vectors containing capn1, capn2, pten and mkp-1 gene (Ad-Capn2, Ad-Capn1, Ad-Pten, Ad-MKP-1, SignaGen, USA) or β-gal (Ad-gal, Vector Biolabs, USA) as a control at a multiplicity of infection of 100 plaque forming units/cell ( Peng et al. 2003 ).

Techniques: Saline, Expressing, Western Blot, Quantitation Assay