Fig 1.
Regulation of hypoxia-inducible proteins and malonyl CoA levels by hypoxia.
(A) Protein expression of hypoxia-inducible proteins, including HIF-1α, aryl hydrocarbon receptor nuclear transport (ARNT), and lactoferrin. (B) Schematic depicting regulation of malonyl CoA levels by acetyl CoA carboxylase (ACC) and malonyl CoA decarboxylase (MCD). Protein expression of ACC and MCD measured by Western blot with quantification determined using densitometry with background subtraction. The contrast and brightness were altered to 46% and -20%, respectively, uniformly throughout the ARNT blot in order to enable increased distinction of the band from the background. The unmodified, uncropped western blots are provided in the supplemental material. Malonyl CoA levels in HCFs and HKCs were measured by ELISA. All data was normalized to HCF-Normoxic control. n≥3, error bars represent SEM. An ANOVA was used to determine statistical significance with *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001.
Fig 2.
Secretion of the major collagens, Col I, III, and V, detected in the media following exposure to normoxic or hypoxic (2% O2) conditions.
(A) Representative western blots and (B-D) quantification by densitometry showing a reduction in Col I in both HCFs and HKCs following hypoxia exposure. Error bars represent standard error of the mean. n = 3 for Col I and III, n = 2 for Col V. A two-way ANOVA was used to determine statistical significance with *p≤0.05, **p≤0.01, ***p≤0.001, and ****p≤0.0001.
Fig 3.
Effects of hypoxia on MMP, collagen expression, and FAK signaling in HCFs and HKCs under normoxic or hypoxic (2% O2) conditions.
(A) Representative western blots and (B-F) quantification for MMP-9, MMP-2, MMP-1, MMP-13, and MMP-3 expression. (G) Representative western blots of cytosolic collagen and keratocan expression and quantification of (H-K) Collagens I, III, V, and keratocan expression. (L) Representative western blot of pFAK and FAK expression and (M) quantification of the ratio of pFAK/FAK. Quantification determined using densitometry. n = 3, statistical significance determined by an ANOVA with * = p≤0.05, ** = p≤0.01, *** = p≤0.001, and **** = p≤0.0001.
Table 1.
ECM thickness after 4 weeks at normoxic conditions or maintained for 3 weeks at normoxia and then transferred to a hypoxia environment (2% O2) for 1 week.
The distance from top to bottom cell layer was measured by confocal microscopy. n = 3, mean±S.E.M. Statistical significance determined by ANOVA, comparing all values to HCF-Normoxic controls.