Figure 5. In vitro volutrauma activates mTORC1 through reactive oxygen species-dependent activation of the ERK pathway.

(A) Human bronchial epithelial cells were subjected to volutrauma (20% equibiaxial stretch, 0.2 Hz, 30 min) in the presence of increasing doses of the ERK 1/2 inhibitor (SCH772984) or vehicle (DMSO) prior to immunoblotting for markers of ERK and mTORC1 activation (pooled protein from n = 2 wells/lane). (B) SAECs were subjected to volutrauma (24% biaxial stretch, 0.2 Hz, 30 min) in the presence of increasing doses of the AKT inhibitor (MK-2206) or vehicle (DMSO) prior to immunoblotting for markers of AKT and mTORC1 activation (pooled protein from n = 2 wells/lane). (C and D) HBEs were stretched (24% biaxial stretch, 0.2 Hz, 30 min) in the presence of CellRox Green and fluorescence was quantitated in each field (100×). Data log normally distributed, analyzed by Student’s t test on log₂ transformed data (n = 18 fields/group). (E and F) SAECs were stretched (24% biaxial stretch, 0.3 Hz, 30 min) in the presence of mitoSOX (Red) and calcein AM (Green) prior to quantitating the intensity of MitoSOX Red staining in each field (100×). Data not normally distributed, analyzed by Mann-Whitney test (n = 27 images per condition). (G) Small airway epithelial cells were treated with 500 μM hydrogen peroxide (H₂O₂) for increasing amounts of time prior to immunoblotting for markers or ERK and mTORC1 activation (n = 1 well/lane). (H) SAECs were treated with increasing doses of GSH-EE 30 minutes prior to volutrauma (20% equibiaxial stretch, 0.2 Hz, 30 min) and immunoblotting for markers of mTORC1 and ERK activation (n = 2 wells/lane). Box blots show median ± interquartile range and whiskers define min and max values. *P < 0.05 for all panels.