Figure 1.
Hypoxia changes cell size and focal contact number.
(A) Hypoxia increases the cell area of L929 fibroblasts. Cells were incubated in normoxic (20% O2) or hypoxic conditions (1% O2) for 24 hrs fixed and stained with phalloidin-FITC. The cell area of single cells was measured and was calculated as fold change compared to 20% O2. (B) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia for 24 hrs. Cells were harvested after 24 hrs. Single cell suspension was prepared by enzymatic digestion. (C) Immunofluorescence images of vinculin in L929 cells. Focal contacts were counted after 24 hrs of hypoxic or normoxic incubation. Note the increase in vinculin positive focal contacts in hypoxia. Arrows point to exemplary focal contacts. (D) Flow cytometry analysis of L929 cells after incubation for 24 hrs in normoxia and hypoxia stained with integrin β1 antibodies. Numbers within the bars indicate the number of cells analysed. ** p<0.01, *** p<0.001. Bars represent mean values±SEM.
Figure 2.
Hypoxia changes cell spreading and migration.
(A) Spreading of L929 cells in normoxia and hypoxia. L929 cells were incubated under normoxic (20% O2) or hypoxic conditions (1% O2), trypsinized and replated for 20 min. Cells were fixed, stained with phalloidin-FITC and divided into three categories (a: round, barely spread; b: in the course of spreading; c: well spread). The percentage of cells in each category was determined. Note that L929 fibroblasts spread faster under hypoxic conditions. (B) Scratch wound healing assays under normoxic and hypoxic conditions. The cell monolayer was scratched with a sterile pipette tip. Images were taken 6, 12, 30 hrs after wounding and the cell-free area was determined. Note that L929 fibroblasts in hypoxia migrate slower into the scratch. (C) Single cell migration of L929 fibroblasts. After 24 hrs of normoxic and hypoxic incubation bright field images of the cells were taken over a period of 360 min. Images were superimposed and the path of the cells was reconstructed by monitoring the cells at each timepoint (going from black to brown). Note that single cell migration is slower under hypoxic conditions. Numbers within the bars indicate the number of cells analysed. * p<0.05, ** p<0.01, *** p<0.001. Bars represent mean values±SEM.
Figure 3.
Hypoxia induces the reorganisation of the actin cytoskeleton.
β- and γ-actin distribution in L929 fibroblasts in normoxia (A) and in hypoxia (B). Cells were grown for 24 hrs in normoxic or hypoxic conditions and subsequently stained for β-actin and γ-actin. Under normoxic conditions both actin isoforms colocalize. In contrast, under hypoxic conditions the cells develop ring-like actin bundles in the cell center, which are greatly enriched with β-actin, while γ-actin is enriched at the cell periphery.
Figure 4.
HIF-1α stabilisation regulates cell area.
(A) Schematic drawing of HIF-1 and its regulation in normoxia and hypoxia. The stability of the HIF-1α subunit is regulated by prolyl-4-hydroxylase domain (PHD) enzymes in an oxygen dependent manner. Following hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognises HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation under normoxic conditions. Hypoxia impairs the hydroxylation, which results in HIF-1α stabilisation, nuclear accumulation, heterodimerisation with HIF-1β and subsequent hypoxia-inducible gene expression. (B) Inhibition of PHDs by DMOG causes HIF-1α stabilisation under normoxic conditions. L929 cells were treated with 1 mM DMOG for 48 hrs and total extracts were analysed by immunoblot with the respective antibodies. (C) The cell area of DMOG treated cells increased significantly. The cell area of single cells was measured and was calculated as fold change compared to 20% O2. (D) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were obtained via stable transduction of specific sh-plasmids. The HIF-1α knock down was verified in western blot experiments of cell lysates from cells incubated at 20% O2 or 1% O2. β-tubulin was used as a loading control. (E) HIF-1α knock down cell clones (c1, c2) do not respond to hypoxia with an increase in cell area. The cell area of single cells was measured and was calculated as fold change compared to the cell clone cultivated at 20% O2. (F) Flow cytometry analysis of cell volume after incubation in normoxia and hypoxia. shC, c1 and c2 cells were harvested after 24 hrs of normoxic (20% O2) or hypoxic (1% O2) incubation. Single cell suspension was prepared by enzymatic digestion. Note that hypoxia increases the cell volume independently of the HIF-1α knock down.
Figure 5.
Depletion of HIF-1α does not affect the number of focal contacts and cell spreading.
(A) Two HIF-1α knock down cell clones (c1, c2) and a non-target control shRNA cell clone (shC) were stained for vinculin 24 hrs after normoxic (20% O2) or hypoxic (1% O2) incubation. Counting vinculin positive focal contacts showed a higher number of focal contacts in hypoxia in all three cell lines. (B) Flow cytometry analysis of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA cell clone (shC) after 24 hrs of normoxia or hypoxia stained with integrin β1 antibodies. (C) Wt, shC, and the HIF-1α knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O2) or hypoxia (1% O2). Cell extracts were analysed by Western blots. Note that vinculin and integrin β1 levels are not changed in hypoxia. (D) Cell spreading of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were incubated at 20% O2 and 1% O2, trypsinised and replated for 20 min. Cells were fixed and stained with phalloidin-FITC and divided into three categories (a: round, barely spread; b: in the course of spreading; c: well spread). The percentage of cells in each category was determined. All three cell lines spread faster under hypoxic conditions. Numbers within the bars indicate the number of cells analysed. ** p<0.01, *** p<0.001. Bars represent mean values±SEM.
Figure 6.
Depletion of HIF-1α alters cell motility.
(A) Wounding assay of the HIF-1α knock down cell clones c1 and c2 and the non-target control shRNA (shC) cell clone in normoxia and hypoxia. Cells were grown in normoxia and hypoxia and experimental wounds were caused by scratching cell monolayers with a pipet tip. Images were taken at the indicated time points and the cell free area was determined. Hypoxia delayed wound healing. Note that knocking down HIF-1α slowed wound closure down even more (dashed red line). (B) Single cell migration of the HIF-1α knock down cell clones c1 and c2 and the shC cells. Cells were incubated at 20% O2 and 1% O2 for 24 hrs. Images were taken over a time period of 360 min and cell movement was analysed. shC cells showed a reduced migration under hypoxic condition, however this effect was not seen in the HIF-1α knock down clones c1 and c2. Numbers within the bars indicate the number of cells analysed. ** p<0.01, *** p<0.001. Bars represent mean values±SEM.
Figure 7.
HIF-1α knock down affects cytoplasmic actin reorganisation in hypoxia.
ShC cells, and the HIF-1α knock down cell clones c1 and c2 cells were incubated at 20% and 1% O2 for 24 hrs and stained for β-actin and γ-actin. Whereas the shC cells display an actin isoform redistribution in hypoxia no obvious actin reorganisation is seen in c1 and c2 cells.
Figure 8.
Cofilin phosphorylation is HIF-1α dependent.
Wt, shC, and the HIF-1α knock down cell clones c1 and c2 cells were lysed after 24 hrs of normoxia (20% O2) or hypoxia (1% O2). Cell extracts were analysed by Western blots. Note that p-cofilin levels are reduced in c2 and c2 cells compared to wt and shC cells at 20% O2 and 1% O2.
Figure 9.
Schematic summary of hypoxia induced and HIF-1α dependent changes in L929 fibroblasts.
Hypoxia induces changes in cell architecture and function. Some of these changes can be linked to stabilisation of HIF-1α.↑, increase; ↓ decrease; ↔, no change when compared to normoxia. HIF-1α dependent effects, which are not seen in hypoxia after HIF-1α knock down, are highlighted in yellow.