Fig 1.
Hypoxia increases the activity of distinct mTORC1 downstream targets in keratinocytes.
(A) Western blot showing total cell lysates of MKs and HaCaT cells exposed to hypoxia and probed for phosphorylated p70S6K (Thr389), p70S6K, phosphorylated 4E-BP1 (Thr70), 4E-BP1, and β-actin (loading control). (B) Quantitative analysis of expression of the aforementioned proteins in MKs. The graphs represent the mean ± SD (n = 3) of the relative integrated signals. N, normoxia. *P< 0.05 versus the N group. (C) Quantitative analysis of the expression of these proteins in HaCaT cells. The graphs represent the mean ± SD (n = 3) of the relative integrated signals. N, normoxia. *P< 0.05 versus the N group.
Fig 2.
mTORC1 is inhibited by rapamycin and shRNA against Raptor.
(A) Keratinocytes were incubated under normoxic and hypoxic conditions in the presence or absence of rapamycin (100 mM, SB) or the shRNA against Raptor (shRaptor). Western blotting was performed to analyze the expression of phosphorylated p70S6K (Thr389), p70S6K, phosphorylated 4E-BP1 (Thr70), 4E-BP1, and β-actin (loading control). (B) The graphs represent the mean ± SD (n = 3) of the relative integrated signals. N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h + DMSO group. (C) The graphs represent the mean ± SD (n = 3) of the relative integrated signals. N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h + shNC group.
Fig 3.
The mTORC1 pathway is required for hypoxia-induced keratinocyte motility and migration.
(A) The movement trajectories of cells with mTORC1 inhibition under normoxic or hypoxic culture condition. (B) Statistical analysis of the trajectory speeds of mTORC1-inhibited MKs and HaCaT keratinocytes. The data were from at least 100 cells in 3 independent experiments and are shown as the mean ± SD. N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h + DMSO group. ##P< 0.05 versus the hypoxia 6 h + shNC group. (C) Wound-healing assays were performed using Culture-Inserts. The cell migration of normoxic and hypoxic MKs with mTORC1 inhibition were recorded for 24 h after wounding. Scalebar = 200 μm. The wound closure (%) was the reduction in the original cell-free wound area. (D) The graph represents the mean ± SD (n = 3). N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h + DMSO group. ##P< 0.05 versus the hypoxia 6 h + shNC group.
Fig 4.
Hypoxia mediates time-dependent, sustained suppression of the AMPK signalling pathway in keratinocytes.
(A) Representative cropped blots of phosphorylated AMPKα (Thr172), AMPK, phosphorylated ACC (Ser79), ACC, and β-actin (loading control) in MKs and HaCaT cells under normoxic and hypoxic conditions. (B) The graphs represent the mean ± SD of the relative integrated signals in MKs. N, normoxia. *P< 0.05 versus the N group. (C) The statistical graphs represent the mean ± SD of the relative integrated signals in HaCaT cells. N, normoxia. *P< 0.05 versus the N group.
Fig 5.
AMPK activation reverses hypoxia-induced mTORC1 activation.
(A) Western blot analysis of the effects of AMPK pathway activators Met and AICAR on the expression of phosphorylated p70S6K (Thr389), p70S6K, phosphorylated 4E-BP1 (Thr70), 4E-BP1, phosphorylated AMPKα (Thr172), AMPK, phosphorylated ACC (Ser79), ACC and β-actin (loading control). (B) The graphs represent the mean ± SD (n = 3) of the relative integrated signals in MKs. N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h group. (C) The statistical graphs represent the mean ± SD (n = 3) of the relative integrated signals in HaCaT cells. N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h group.
Fig 6.
AMPK is involved in keratinocyte migration under hypoxia.
(A) The representative migration trajectories of MKs under normoxic or hypoxic conditions in the presence or absence of Met and AICAR. (B) Statistical analysis of the trajectory speeds of AMPK-activated MKs and HaCaT cells. The data were from at least 100 cells in 3 independent experiments and were shown as the mean ± SD. N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h group. (C) Cell migration was measured by using Culture-Inserts. The wound closure (%) was the reduction in the original non-covered wound area. Scalebar = 100 μm. (D) The panels represent the covered wound areas and show the mean ± SD (n = 3). N, normoxia. *P< 0.05 versus the N group. #P< 0.05 versus the hypoxia 6 h group.