Ished by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the Animal Care and Use Committee of the Renmin Hospital of Wuhan University (protocol number: 00020390).Male KO mice(C57BL/6 background; 22948146 knockout mice were purchased from Jackson Laboratory, No. 006908) and their wild-type (WT) littermates, which ranged in age from 8 to 10 weeks, were subjected to aortic banding (AB) as described previously [27]. Each mouse was were anaesthetized with sodium pentobarbital (Sigma, 80 mg/kg, ip), and a horizontal skin incision was made at the level of 2? intercostal space. The descending aorta was isolated, and a 7.0 silk suture was wrapped around the aorta.A bent 26-gauge needle (for 25.5?7.5 g) or 27gauge needle (for 23.5?5.5 g) was then placed next to the aorta, and the suture was tied snugly around the needle and the aorta. After ligation, the needle was quickly removed, the chest and skin were closed, and the mice were allowed to recover. Sham-operated mice underwent the same procedure without constriction. The adequacy of anesthesia was monitored during the surgical procedures by assessing the lack of the pedal withdrawal reflex, slow constant breathing, and no response to surgical manipulation. Buprenorphine (0.1 mg/kg, sc) was administered for post-operative analgesia. Four weeks after the operation, the hearts, lungs, and tibiae of the mice were dissected and weighed or measured to compare the heart weight (HW)/body weight (BW) (mg/g), HW/ tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios of the different groups.Quantitative real-time RT-PCRReal-time PCR was performed to detect the mRNA expression levels of hypertrophic and fibrotic markers. Total RNA was PD-168393 site extracted from frozen pulverized mouse cardiac tissue using TRIzol (Invitrogen, 15596-026). The yields and purities were spectrophotometrically estimated using the A260/A280 and A230/260 ratios, as measured with a SmartSpec Plus Spectrophotometer (Bio-Rad). The RNA (2 mg of each sample) was reverse-transcribed into cDNA using oligo(DT) primers and the buy SR3029 Transcriptor First Strand cDNA Synthesis Kit (Roche, 04896866001). The PCR amplifications were quantified using the LightCycler 480 SYBR Green 1 Master Mix (Roche,04707516001). The results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression.Protein extraction and western blotting analysesThe left ventricles were harvested for western blotting analyses. They were first lysed in RIPA lysis buffer, and the protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 
23227) and an ELISA Reader(Synergy HT, Bio-Tek). The cell lysates (50 mg) were loaded into each lane and subjected to SDS-PAGE, and the proteins were then transferred onto Immobilon-FL transfer membranes (Millipore, IPFL00010). The membranes were incubated overnight at 4uC with primary antibodies against one of the following proteins: p-MEK1/2 (Cat#, 9154), T-MEK1/2 (Cat#,9122), p-ERK1/2 (Cat#,4370), T-ERK1/2 (Cat#,4695), p-P38 (Cat#,4511), T-P38 (Cat#,9212), p-JNK(Cat#,4668),T-JNK (Cat#,9258), p-PI3K(Cat#, 4228), TPI3K (Cat#,4257), p-AKT (Cat#,4060), T-AKT (Cat#,4691), PGSK3b (Cat#,9322), T-GSK3b (Cat#,9315), p-mTOR (Cat#,2971), T-mTOR (Cat#,2983), P-FOXO3A (Cat#,9465), T-FOXO3A (Cat#,2497), P-FOXO1 (Cat#,9461), T-FOXO1 (Cat#,2880), p-NFkB (Cat#,3033), T-NFkB (Cat#,4764), Bax (Cat#,2772), Bcl2(Cat#, 2870),Cleaved Caspase3(Cat#,9661),GAPDH(Cat#,2118),and IKKi(Cat#,3.Ished by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the Animal Care and Use Committee of the Renmin Hospital of Wuhan University (protocol number: 00020390).Male KO mice(C57BL/6 background; 22948146 knockout mice were purchased from Jackson Laboratory, No. 006908) and their wild-type (WT) littermates, which ranged in age from 8 to 10 weeks, were subjected to aortic banding (AB) as described previously [27]. Each mouse was were anaesthetized with sodium pentobarbital (Sigma, 80 mg/kg, ip), and a horizontal skin incision was made at the level of 2? intercostal space. The descending aorta was isolated, and a 7.0 silk suture was wrapped around the aorta.A bent 26-gauge needle (for 25.5?7.5 g) or 27gauge needle (for 23.5?5.5 g) was then placed next to the aorta, and the suture was tied snugly around the needle and the aorta. After ligation, the needle was quickly removed, the chest and skin 
were closed, and the mice were allowed to recover. Sham-operated mice underwent the same procedure without constriction. The adequacy of anesthesia was monitored during the surgical procedures by assessing the lack of the pedal withdrawal reflex, slow constant breathing, and no response to surgical manipulation. Buprenorphine (0.1 mg/kg, sc) was administered for post-operative analgesia. Four weeks after the operation, the hearts, lungs, and tibiae of the mice were dissected and weighed or measured to compare the heart weight (HW)/body weight (BW) (mg/g), HW/ tibial length (TL) (mg/mm), and lung weight (LW)/BW (mg/g) ratios of the different groups.Quantitative real-time RT-PCRReal-time PCR was performed to detect the mRNA expression levels of hypertrophic and fibrotic markers. Total RNA was extracted from frozen pulverized mouse cardiac tissue using TRIzol (Invitrogen, 15596-026). The yields and purities were spectrophotometrically estimated using the A260/A280 and A230/260 ratios, as measured with a SmartSpec Plus Spectrophotometer (Bio-Rad). The RNA (2 mg of each sample) was reverse-transcribed into cDNA using oligo(DT) primers and the Transcriptor First Strand cDNA Synthesis Kit (Roche, 04896866001). The PCR amplifications were quantified using the LightCycler 480 SYBR Green 1 Master Mix (Roche,04707516001). The results were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression.Protein extraction and western blotting analysesThe left ventricles were harvested for western blotting analyses. They were first lysed in RIPA lysis buffer, and the protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 23227) and an ELISA Reader(Synergy HT, Bio-Tek). The cell lysates (50 mg) were loaded into each lane and subjected to SDS-PAGE, and the proteins were then transferred onto Immobilon-FL transfer membranes (Millipore, IPFL00010). The membranes were incubated overnight at 4uC with primary antibodies against one of the following proteins: p-MEK1/2 (Cat#, 9154), T-MEK1/2 (Cat#,9122), p-ERK1/2 (Cat#,4370), T-ERK1/2 (Cat#,4695), p-P38 (Cat#,4511), T-P38 (Cat#,9212), p-JNK(Cat#,4668),T-JNK (Cat#,9258), p-PI3K(Cat#, 4228), TPI3K (Cat#,4257), p-AKT (Cat#,4060), T-AKT (Cat#,4691), PGSK3b (Cat#,9322), T-GSK3b (Cat#,9315), p-mTOR (Cat#,2971), T-mTOR (Cat#,2983), P-FOXO3A (Cat#,9465), T-FOXO3A (Cat#,2497), P-FOXO1 (Cat#,9461), T-FOXO1 (Cat#,2880), p-NFkB (Cat#,3033), T-NFkB (Cat#,4764), Bax (Cat#,2772), Bcl2(Cat#, 2870),Cleaved Caspase3(Cat#,9661),GAPDH(Cat#,2118),and IKKi(Cat#,3.
