1. Fig. 3. Western blot analysis of cell cycle regulators reveals loss of at least one CDK inhibitor. 75 ug/lane of protein extracts were loaded onto 8% (for HIF-1 and actin) or 12% acrylamide SDS–PAGE gels and transferred to the appropriate membrane. Pan actin blots were performed for all cell lines to normalize protein-loading amounts. ( A ) HTB-30 cells lines exhibited a loss of p16 protein, similar to WI-38 normal fibroblasts (previous figure) and also exhibited loss of another INK4 family member, p19. HTB-30 also showed an induction of p27 protein levels late in the hypoxic response. ( B ) Hep3B exhibited an induction of p27 late in the hypoxic response, but also showed loss of p19 and p21, similar to the observed reduction in the HTB-30 cell line. Hep3B p16 levels, however, were invariant in hypoxia. ( C ) HeLa cells showed reductions in all CDKIs measured, including p27, although they still underwent hypoxia-induced cell cycle arrest. ( D ) Primary breast epithelial cells (HMEC) showed dramatic reductions in p21 levels and p16, starting ∼12 h after initial exposure to hypoxia. Other CDKI levels (p15, p18, p19) were below detection thresholds in this cell strain.
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.

2. Fig. 1. Cell cycle analysis of selected cell lines in hypoxia
Fig. 1. Cell cycle analysis of selected cell lines in hypoxia. Cells were incubated in a normoxic (20% O ₂ ; UT) or hypoxic (<0.5% O ₂ ; T) atmosphere for 2 or 24 h, fixed in ethanol, stained with propidium iodide and analysed for DNA content to determine G ₁ and S phase cell populations. ( A ) Graphical representation of flow cytometric analysis of the HTB-30 cell line shows an increase in G ₀ /G ₁ and a decrease in S phase, indicating cell cycle arrest in response to hypoxia. ( B ) The HeLa cell line also shows hypoxia-induced cell cycle arrest. ( C ) Hep3B cells do not exhibit a significant increase in G ₀ /G ₁ or decrease in S phase, indicating a lack of cell cycle arrest, even after 24 h of hypoxia. ( D ) Flow cytometry histograms of primary HMEC cells show a gradual decrease in S phase and M phase starting at ∼6 to 12 h after initial exposure to low O ₂ , with a parallel increase in percentage of cells in G ₀ /G ₁ . ( E ) Primary WI38 fibroblasts exhibit a decrease in cells transiting through S phase.
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.

3. Fig. 2. p16 mRNA levels are reduced in diploid fibroblasts WI-38
Fig. 2. p16 mRNA levels are reduced in diploid fibroblasts WI-38. p16 mRNA levels were analysed by RPA or northern blot in cell lines incubated in a normoxic (20% O ₂ ; UT) or hypoxic (<0.5% O ₂ ; T) atmosphere for the times indicated. Levels of L32 transcript were used as a loading control for RPA and 28S rRNA was used in northern blot analysis. Identically treated whole cell lysates were prepared for immunoblot analysis. ( A ) WI-38 fibroblast cells, which undergo cell cycle arrest, reveal a reduction in p16 mRNA levels starting at 12 h (12H T) after exposure to hypoxia, as measured by RPA. ( B ) Northern blots of p16 mRNA levels confirm p16 mRNA reductions in WI-38 fibroblasts treated with hypoxia for 24 h (24H T). ( C ) p16 mRNA levels correlate with a reduction in p16 protein levels in WI-38 fibroblasts. ( D ) HMEC and HTB-30 cell lines exhibit a similar reduction in p16 mRNA levels, as measured by RPA, while L32 ribosomal protein mRNA loading standards are unchanged.
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.

4. Fig. 4. Western blots indicate a lack of PARP cleavage during hypoxia time course. Cell lines were incubated in a normoxic (20% O ₂ ; UT) or hypoxic (<0.5% O ₂ ; T) atmosphere for the times indicated and whole cell lysates were prepared for immunoblot analysis. Pan actin protein levels were used as a protein loading control. Analysis of PARP in HeLa, Hep3B, HTB-30 and HMEC cells showed that PARP was maintained at its full-length size of ∼119 kDa, indicating a lack of caspase activity and subsequent apoptosis. No faster migrating versions of PARP were detected.
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.

5. Fig. 5. Phospho-specific western blots reveal differential AKT/GSK3β activation in cells with and without an intact hypoxia-induced G ₁ /S arrest. Whole cell lysates were prepared from cells incubated in normoxia (20% O ₂ ; UT) or hypoxic (<0.5% O ₂ ; T) atmosphere for 2, 6, 12 and 24 h. Western blotting of Akt and GSK3β showed that in hypoxia-treated HeLa cells ( A ), Akt exhibited activating phosphorylation, but phosphoprotein and total protein levels were lost between 6 and 12 h post-hypoxia. Concomitantly, inhibitory phosphorylation on GSK3β was lost. ( B ) Hep3B cells did not exhibit modulation of Akt and GSK3β phosphorylation with hypoxia treatment.
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.

6. Fig. 6. Inhibition of GSK3β activity prevents degradation of HIF-1 and CDKI proteins in hypoxia. HeLa cells were pre-treated with 50 mM LiCl (final), added directly to the tissue culture media, immediately prior to incubation in a normoxic (20% O ₂ ; UT) or hypoxic (<0.5% O ₂ ; T) atmosphere for the times indicated (2, 6, 12 and 24 h). Whole cell lysates were prepared and protein levels were determined by immunoblotting. Pan actin levels were used to control for total protein loading. ( A ) Normoxic HeLa cells were treated in the presence or absence of 50 mM LiCl for 12 or 24 h. Cell extracts were electrophoresed, blotted and detected with antibodies to HIF-1α, p16, p27 and actin. ( B ) Hypoxic (T) or normoxic (UT) HeLa cells were treated in the presence of 50 mM LiCl for 12 or 24 h. Cell extracts were electrophoresed, blotted and detected with antibodies to HIF-1α, p16, p27 and actin. Over time, no decrease in HIF-1α, p16or p27 was noted in the hypoxic cells treated with LiCl, in sharp contrast to their expression when LiCl was not present ( Figure 3 ).
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.

7. Fig. 7. A proposed model outlining an Akt/GSK3β-dependent mechanism for INK4 and Cip/Kip down-regulation late in the hypoxic response.
From: Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines
Carcinogenesis. 2004;25(12): doi: /carcin/bgh274
Carcinogenesis | Carcinogenesis vol.25 no.12 © Oxford University Press 2004; all rights reserved.