1. Hypoxia Impairs Skin Myofibroblast Differentiation and Function
Ali Modarressi, Giorgio Pietramaggiori, Charles Godbout, Enrico Vigato, Brigitte Pittet, Boris Hinz 
Journal of Investigative Dermatology 
Volume 130, Issue 12, Pages (December 2010)
DOI: /jid
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Switching to low O2 affects cultured rat subcutaneous fibroblasts (SCFs). SCFs were grown for 5 days in 21% O2 (standard culture), and 10, 5, and 2% O2. (a) Expression of α-smooth muscle actin (α-SMA) and hypoxia-inducible transcription factor-1α (HIF-1α) assessed by western blot, normalized to housekeeping vimentin for densitometric band analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as additional loading control. (b) Cell number was quantified by cell counting; viability was assessed using a colorimetric cell viability assay, and apoptosis with a caspase-2/3 activity kit. Viability and apoptosis were compared between similar cell numbers at the time of the test. Mean values were calculated from at least three independent experiments performed with five samples per condition. Immunofluorescence staining of SCFs in (c) 21% high O2 and (d) 2% low O2 for α-SMA (red), F-actin (phalloidin, green), and nuclei (blue) was used to quantify the percentage of α-SMA-positive myofibroblasts by automated image analysis (e). Scale bar=20μm. Error bars indicate SD of mean. *P<0.01 tested by Student's t-tests against control 21% O2.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Effect of low O2 on subcutaneous fibroblast (SCF) contraction. Rat SCFs were grown for 5 days in O2 concentrations of (a) 21% and (b) 2% on wrinkling silicone substrates and observed in phase-contrast microscopy. Substrate stiffness restricts formation of surface wrinkles (arrowheads) to highly contractile myofibroblasts. Scale bar=20μm. (c) The percentages of wrinkling SCFs in 21% O2 and 2% O2 were quantified. (d) Contraction of SCF populations was assessed using attached collagen lattices. A minimum of five lattices were assayed per experimental condition, lattice diameter reduction was normalized to the lattice diameter before release (percent contraction), and mean values were calculated from at least three independent experiments. Error bars indicate SD of mean. *P<0.01 tested by Student's t-tests against 21% O2.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Tissue-specific fibroblast reaction to low O2. Primary explant cultures of rat fibroblasts from (a) lung, (b) liver, and (c) heart were grown for 5 days in 21% O2 (top) and 2% O2 (bottom). Cells were then immunostained for α-smooth muscle actin (α-SMA, red), F-actin (phalloidin, green), and nuclei (blue) (a–c) and processed for (d) western blotting. Scale bar=50μm.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
The effect of low O2 on subcutaneous fibroblasts (SCFs) in the presence of active transforming growth factor-β1 (TGFβ1). (a) The levels of active and total TGFβ1 in medium conditioned by SCFs grown for 5 days in 21% and in 2% O2 were determined using transformed mink lung epithelial cells (TMLCs) as reporter cells. Data are presented as the mean of one representative experiment of at least five independent experiments. TGFβ1 (2ngml−1 ) was added to SCFs grown for 5 days in (b) 21% O2 and (c) 2% O2. Cells were then immunostained for α-smooth muscle actin (α-SMA, red), F-actin (green), and nuclei (blue) and (d) the percentage of α-SMA-positive cells was quantified. (e) SCFs extracted from the same conditions were tested by western blotting with loading controls vimentin, α-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); box includes TGFβ1-treated conditions. Contraction of TGFβ1-treated SCFs in (f) 21% and (g) 2% O2 was assessed by growth on silicone substrates that wrinkle only on high myofibroblast contraction. (h) Wrinkling and collagen contraction by TGFβ1-treated SCFs is normalized to 21% O2 without TGFβ1 addition. Scale bar=50μm. Error bars indicate SD of mean. *P<0.01 tested by Student's t-tests against 21% O2.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Correlation of subcutaneous fibroblast (SCFs) contraction and myofibroblast differentiation over culture time in low O2. (a) Rat SCFs were grown in 21% O2 for 5 days minus the indicated times for which the culture was transferred to 2% O2. For the last 6 hours in 2% O2, SCFs were subcultured on soft wrinkling silicone substrates; “0h” cells were always kept in 21% O2. After 6 hours growth on silicone substrates, cells were immunostained for α-smooth muscle actin (α-SMA, red), F-actin (phalloidin, green), and nuclei (blue) (top panel) and overlaid with phase-contrast (wrinkle) images (bottom panel). Note that the soft substrates used here permit even low-contractile fibroblasts to produce wrinkles, in contrast to the stiff substrates used in Figures 2 and 4. (b) Immunostaining and phase-contrast images of wrinkling substrates were separately analyzed for the percentages of wrinkling cells and α-SMA-positive cells of all cells over time in 2% O2 or (c) to determine the percentages of wrinkling cells of all α-SMA-positive cells (white columns) and of all α-SMA-negative cells (gray columns). (d) SCFs extracted from the same conditions were tested by western blotting. Scale bar=100μm. Error bars indicate SD of mean. *P<0.01 tested by Student's t-tests against 21% O2 in each experimental group.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Reversibility of low-O2-induced effects by O2 elevation and mechanical stimulation. Rat subcutaneous fibroblasts (SCFs) were grown for 5 days in 2% O2, followed by another 5 days in (a) 2% O2, (b) 21% O2, (c) 2% O2 under cyclic stretch of 10% with a frequency of Hz, and (d) 21% O2 under cyclic stretch. Cells were immunostained for α-smooth muscle actin (α-SMA, red), F-actin (green), and nuclei (blue). (e) SCFs were assessed for percentages of α-SMA-positive cells and of myofibroblasts wrinkling stiff silicone substrates. Collagen gel contraction was assessed after 3 days of growth under the same O2 conditions as used for the “rescue.” Percentage values are calculated using 2 × 5 days 21% O2 culture as reference (dotted line) (5+3 days in the case of collagen contraction). (f) Western blots were performed with cells extracted after each treatment. Scale bar=100μm. Error bars indicate SD of mean. *P<0.01 tested by Student's t-tests against 2 × 5 days (collagen: 5+3 days) 21% O2 culture in each experimental group.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions