Fig 1.
Time course profile of cell viability, autophagy and apoptosis in TG-induced ER stress with/without addition of autophagy activator/inhibitor.
A) HEK293T cells were treated with 10 μM TG for two hours and pre-treated with rapamycin (100 nM for two hours) or 3-MA (1 mM for two hours) followed by TG addition (10 μM for two hours), meanwhile the relative number of viable cells was denoted in time (error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ∗—p < 0.05; ∗∗—p < 0.01). B) During TG treatment the markers of autophagy (LC3), apoptosis (proCaspase3, PARP), AMPK activation (ULK-555P) and mTOR activation (4-EBP1P, p70SP), as well as GADD34 were followed in time by immunoblotting. During pre-treatmetn with C) 3-MA and D) rapamycin followed by TG addition the autophagy (LC3), the apoptosis (PARP), the AMPK (ULK-555P) and the mTOR (4-EBP1P) markers and GADD34 were followed in time by immunoblotting. GAPDH was used as loading control.
Fig 2.
Time course profile of cell viability, autophagy and apoptosis in TG-induced ER stress when GADD34 was inhibited.
HEK293T cells were pre-treated with GB (5 μM for one hour) followed by TG addition (10 μM for two hours). The GB level was kept high until end of the cell treatment. A) The relative number of viable cells after TG treatment was denoted in time (error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ∗—p < 0.05; ∗∗—p < 0.01). B) Markers for autophagy (LC3), apoptosis (PARP), AMPK activation (ULK-555P) and mTOR activation (4-EBP1P) as well as eiF2αP were followed in time by immunoblotting. GAPDH was used as loading control.
Fig 3.
Time course profile of cell viability, autophagy and apoptosis in TG-induced ER stress when both GADD34 and mTOR was inhibited.
HEK293T cells were pre-treated with GB (5 μM for one hour) then with rapamycin (100 nM for two hours) followed by TG addition (10 μM for two hours). The GB level was kept high until end of the cell treatment. A) The relative number of viable cells after TG treatment was denoted in time (error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ∗—p < 0.05; ∗∗—p < 0.01). B) The autophagy (LC3), the apoptosis (PARP), the AMPK (ULK-555P) and the mTOR (4-EBP1P) markers and eiF2αP were followed in time by immunoblotting. GAPDH was used as loading control.
Fig 4.
Time course profile of cell viability, autophagy and apoptosis in TG-induced ER stress when autophagy was hyper-activated by resveratrol.
HEK293T cells were pre-treated with resveratrol (10 μM for twenty-four hours) followed by TG addition (10 μM for two hours). A) The relative number of viable cells after TG treatment was denoted in time (error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ∗—p < 0.05; ∗∗—p < 0.01). B) The relative cell viability was plotted in time after TG treatment (error bars represent standard deviation). C) The autophagy (LC3), the apoptosis (proCaspase3, PARP), the AMPK (ULK-555P) and the mTOR (4-EBP1P, p70SP) markers and GADD34 were followed in time by immunoblotting. GAPDH was used as loading control.
Fig 5.
Time course profile of cell viability, autophagy and apoptosis in TG-induced ER stress when GADD34 inhibition is combined with resveratrol addition.
HEK293T cells were pre-treated with GB (5 μM for one hours) then with resveratrol (10 μM for twenty-four hours) followed by TG addition (10 μM for two hours). The GB level was kept high until end of the cell treatment. A) The relative number of viable cells after TG treatment was represented in time (error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ∗—p < 0.05; ∗∗—p < 0.01). B) The autophagy (LC3), the apoptosis (PARP), the AMPK (ULK-555P) and the mTOR (4-EBP1P) markers and eiF2αP were followed in time by immunoblotting. GAPDH was used as loading control.
Fig 6.
GADD34 down-regulates mTOR pathway during TG-induced ER stress.
GADD34 was silenced in HEK293T cells, then cells were treated with 10 μM TG for two hours and pre-treated with resveratrol (10 μM for twenty-four hours) followed by TG addition (10 μM for two hours). A) The relative number of viable cells after TG treatment was represented in time (error bars represent standard deviation, asterisks indicate statistically significant difference from the control: ∗—p < 0.05; ∗∗—p < 0.01). B) The autophagy (LC3), the apoptosis (PARP), the AMPK (ULK-555P) and the mTOR (4-EBP1P) markers and GADD34 were followed in time by immunoblotting. GAPDH was used as loading control. C) The wiring diagram of the control network with respect to ER stress. The mTOR, the autophagy inducer, the apoptosis inducer and the ER stress sensors are denoted by isolated blue, green, red and orange boxes, respectively. Dashed line shows how the components can influence each other, while blocked end lines denote inhibition.