AMPK

Cell viabilities with increasing concentrations of cisplatin (CP) and doxorubicin (DOXO) under normoxic and hypoxic condition for 48 hours were determined by MTT assay. IC50 values are presented as the means ?SDs (n=4) and * denotes p<0.05.

AMPK inhibition by compound C(Dorsomorphin) inhibited autophagy activation and neuroprotection induced by IPC in PC12 cells. (A) Compound C (Comp C) abolished IPC induced neuroprotection in PC12 cells. Cells were incubated with compound C 5 μM 60 min before the onset of IPC. Twelve hours after IPC, the cells were subjected to OGD for 10 h and cell viability was examined with CCK-8 kit. (B) Compound C reduced LC3II/LC3I ratio. The cells were incubated with compound C 5 μM 60 min before the onset of IPC. Then the cells were harvested 12 h after IPC and subjected to Western blot analysis. Bar represents mean ± SD, n = 3. *P < 0.05, ***P < 0.001 compared with the control group; $$$ P < 0.001 compared with the OGD group; %%% P < 0.001 compared with the IPC + OGD group; # P < 0.05 compared with the IPC group.

The AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, could inhibit the APN-induced overexpression of CXCL1 and CXCL8 and h-JBMMSCs chemotaxis. After treated with WZ4003 and SB203580, the expression of CXCL1 (A and B) and CXCL8 (C and D) in the cells and supernatants of h-JBMMSCs treated with or without APN was evaluated by the ELISA assay (n = 3; *P < 0.05; **P < 0.01)

FGF21 activates myogenic and aerobic myofiber-associated genes expression via AMPK pathway. The activator (acadesine) or inhibitor (dorsomorphin) of AMPK pathway were used to treat the pcDNA3.1-21 or control transfected C2C12 myoblasts. For this experiment, four groups were set up: FGF21-ACA (pcDNA3.1-21‡acadesine), Control-ACA (control‡acadesine), FGF21-DOR (pcDNA3.1-21‡dorsomorphin), and Control-DOR(control‡dorsomorphin). The qRT-PCR was performed to detect the genes expression of FGF21 (A), AMPK (B), Sirt 1, Myoglobin(C), Desmin (D), MEF2c (E), a-actin (F). (G) The C2C12 myoblasts were transiently transfected with pCDNA3.1-21 or pCDNA3.1, Western blot showed FGF21 activated AMPK signal via FGF21-Sirt1-AMPK. For the phosphorylated AMPK (right), the intensity of band was normalized total AMPK, and then normalized by control. (H and I) FGF21, as well as AMPK activator acadesine (ACA), increased phosphorylation of AMPM, and myogenic genes expression, especially MyHC I, which was activated by ACA (FGF21‡ACA) and suppressed by AMPK inhibitor DOR(FGF21‡DOR). The data are presented as mean±SD (*P<0.05, **P<0.01, and P<0.001), n=ˆ3.

Effect of nilotinib on Bcr-Abl kinase activity in ABCB1- and ABCG2- overexpressing CD34⁺CD38^- cells. K562 parental cells and CD34⁺CD38^- subpopulation isolated from K562 cells were treated with nilotinib at 0.01, 0.1 and 1.0 umol/L for 12 h. Equal amount of protein was loaded for western blot analysis as described in the Experimental section. The experiments were repeated at least three times independently, and a representative experiment is shown.

Cells were treated with CP, DOXO, SRT1720 (100 nM), EX527 (100 nM), A769662 (10 μM) and Compound C (1 μM) under normoxic or hypoxic conditions for 48 hours, and then their viabilities were measured by MTT.

Cultured hepatocytes were treated with 10 μM Compound C (Comp.C) for 30 min before treatment with 10 μM SAL, 1 mM AICAR for 3 h. Cell lysates were immunoblotted for the phosphorylation of AMPK, ACC, Akt and GSK3β. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus SAL alone; †P < 0.05, ††P < 0.01 versus AICAR alone. Values are means ± SEM (n = 4).

Cellular senescence and SIRT1 phosphorylation was monitored in PAECs (P2) incubated in DMEM containing HDL (50 mg/L), LDL (50 mg/L), or resveratrol (100 μM) for 24 hours.

Cell viabilities with increasing concentrations of cisplatin (CP) and doxorubicin (DOXO) under normoxic and hypoxic condition for 48 hours were determined by MTT assay. IC50 values are presented as the means ?SDs (n=4) and * denotes p<0.05.

AMPK inhibition by compound C(Dorsomorphin) inhibited autophagy activation and neuroprotection induced by IPC in PC12 cells. (A) Compound C (Comp C) abolished IPC induced neuroprotection in PC12 cells. Cells were incubated with compound C 5 μM 60 min before the onset of IPC. Twelve hours after IPC, the cells were subjected to OGD for 10 h and cell viability was examined with CCK-8 kit. (B) Compound C reduced LC3II/LC3I ratio. The cells were incubated with compound C 5 μM 60 min before the onset of IPC. Then the cells were harvested 12 h after IPC and subjected to Western blot analysis. Bar represents mean ± SD, n = 3. *P < 0.05, ***P < 0.001 compared with the control group; $$$ P < 0.001 compared with the OGD group; %%% P < 0.001 compared with the IPC + OGD group; # P < 0.05 compared with the IPC group.

The AMPK inhibitor, WZ4003, and the p38 MAPK inhibitor, SB203580, could inhibit the APN-induced overexpression of CXCL1 and CXCL8 and h-JBMMSCs chemotaxis. After treated with WZ4003 and SB203580, the expression of CXCL1 (A and B) and CXCL8 (C and D) in the cells and supernatants of h-JBMMSCs treated with or without APN was evaluated by the ELISA assay (n = 3; *P < 0.05; **P < 0.01)

FGF21 activates myogenic and aerobic myofiber-associated genes expression via AMPK pathway. The activator (acadesine) or inhibitor (dorsomorphin) of AMPK pathway were used to treat the pcDNA3.1-21 or control transfected C2C12 myoblasts. For this experiment, four groups were set up: FGF21-ACA (pcDNA3.1-21‡acadesine), Control-ACA (control‡acadesine), FGF21-DOR (pcDNA3.1-21‡dorsomorphin), and Control-DOR(control‡dorsomorphin). The qRT-PCR was performed to detect the genes expression of FGF21 (A), AMPK (B), Sirt 1, Myoglobin(C), Desmin (D), MEF2c (E), a-actin (F). (G) The C2C12 myoblasts were transiently transfected with pCDNA3.1-21 or pCDNA3.1, Western blot showed FGF21 activated AMPK signal via FGF21-Sirt1-AMPK. For the phosphorylated AMPK (right), the intensity of band was normalized total AMPK, and then normalized by control. (H and I) FGF21, as well as AMPK activator acadesine (ACA), increased phosphorylation of AMPM, and myogenic genes expression, especially MyHC I, which was activated by ACA (FGF21‡ACA) and suppressed by AMPK inhibitor DOR(FGF21‡DOR). The data are presented as mean±SD (*P<0.05, **P<0.01, and P<0.001), n=ˆ3.

Effect of nilotinib on Bcr-Abl kinase activity in ABCB1- and ABCG2- overexpressing CD34⁺CD38^- cells. K562 parental cells and CD34⁺CD38^- subpopulation isolated from K562 cells were treated with nilotinib at 0.01, 0.1 and 1.0 umol/L for 12 h. Equal amount of protein was loaded for western blot analysis as described in the Experimental section. The experiments were repeated at least three times independently, and a representative experiment is shown.

Cells were treated with CP, DOXO, SRT1720 (100 nM), EX527 (100 nM), A769662 (10 μM) and Compound C (1 μM) under normoxic or hypoxic conditions for 48 hours, and then their viabilities were measured by MTT.

Cultured hepatocytes were treated with 10 μM Compound C (Comp.C) for 30 min before treatment with 10 μM SAL, 1 mM AICAR for 3 h. Cell lysates were immunoblotted for the phosphorylation of AMPK, ACC, Akt and GSK3β. *P < 0.05, **P < 0.01 versus control; ##P < 0.01 versus SAL alone; †P < 0.05, ††P < 0.01 versus AICAR alone. Values are means ± SEM (n = 4).

Cellular senescence and SIRT1 phosphorylation was monitored in PAECs (P2) incubated in DMEM containing HDL (50 mg/L), LDL (50 mg/L), or resveratrol (100 μM) for 24 hours.