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Figures
- Fig. 1.
An immunoblot showing three HIF-1α forms and individual variation of HIF-1α protein amounts in crucian carp. In the top panel, each lane represents HIF-1α detected in nuclear extracts of liver from normoxic fish at 8°C. The bottom panel shows the gel stained with Coomassie Brilliant Blue.
- Fig. 2.
Cold body temperature activates HIF-1. (A) HIF-1α mRNA amounts, (B) HIF-1α protein amounts, (C) HIF-1 DNA-binding activities in normoxic fish acclimated to 26°C, 18°C or 8°C. Results are shown as fold increase compared to fish acclimated to 26°C and are means ± s.e.m. of 10 fish. HIF-1α protein data includes variation caused by differences in fish mass. Asterisks denote significant (P<0.05, P<0.001, P<0.001, P<0.05 and P<0.01 for heart 18°C, heart 8°C, gills 8°C, kidney 18°C and kidney 8°C, respectively) differences to fish acclimated to 26°C (two-way ANOVA followed by Holm–Sidak post test). (D) Representative electrophoretic mobility shift assays showing binding of HIF-1 on HRE of human erythropoietin gene in nuclear extracts from gills of fish acclimated to 26°C, 18°C or 8°C (5 individuals for each temperature).
- Fig. 3.
Cold temperature induces heat shock proteins. (A) Hsp90 amounts in normoxic fish at 26°C, 18°C or 8°C. Results are shown as fold increase compared to levels in normoxic fish at 26°C and are means ± s.e.m. of 5–10 fish. Asterisks denote significant (P<0.001) differences to fish at 26°C (ANOVA and Dunnet's). (B) Hsp70 amounts in normoxic fish at 26°C, 18°C or 8°C. Results are shown as fold increase compared to levels in normoxic fish at 26°C and are means± s.e.m. of 5–10 fish. Asterisks denote significant differences to fish at 26°C (ANOVA and Dunnet's, P<0.001). (C) Interaction of Hsp90 and Hsp70 with HIF-1α in crucian carp. Nuclear extracts from the liver of normoxic crucian carp were immunoprecipitated (IP) with Hsp90 antibody, thereafter HIF-1α, Hsp70 and Hsp90 were detected from the precipitates by immunoblot analysis.
- Fig. 4.
HIF-1α protein amounts in the liver (A), heart (B), gills (C), and kidney (D), of fish exposed to hypoxia at 26, 18 or 8°C. Results are shown as fold increase compared to levels in normoxic fish at 26°C and are means± s.e.m. of 10 fish (includes variation caused by differences in fish mass). Asterisks denote significant effect of hypoxia on HIF-1α amounts (GENMOD, *P<0.01, **P<0.001, ***P<0.0001). †Significant effect of temperature on HIF-1α amounts (GENMOD, P<0.0001). GENMOD analysis does not allow post tests for testing differences between study groups.
- Fig. 5.
HIF-1 DNA-binding activities in the liver (A), heart (B), gills (C), and kidney (D), of fish exposed to hypoxia at 26°C, 18°C or 8°C. Results are shown as fold increase compared to levels in normoxic fish at 26°C and are means ±s.e.m. of 10 fish. Temperature has a significant effect on hypoxic activation of HIF-1 in the heart (P<0.05), gills (P<0.01) and kidney (P<0.001) of crucian carp (two-way ANOVA). *Significant difference to normoxic fish at 26°C (for P-values, see Fig. 2), †significant difference (P<0.05, P<0.01 and P<0.05 for heart 6 h hypoxia, heart 24 h hypoxia and gills 48 h hypoxia, respectively) to normoxic fish at the same temperature, ††significant difference (P<0.05, P<0.05 and P<0.001 for heart, gills and kidney, respectively) to fish exposed to hypoxia for 24 h at the same temperature (Holm–Sidak post test).
- Fig. 6.
HIF-1α mRNA amounts in the liver (A), gills (B) and kidney (C) of fish exposed to hypoxia at 26°C, 18°C or 8°C. Results are shown as fold increase compared to levels in normoxic fish at 26°C and are means± s.e.m. of 5–10 fish. Temperature has a significant effect on the hypoxia response of HIF-1α mRNA in the liver (P<0.05), gills (P<0.01) and kidney (P<0.01) of crucian carp (two-way ANOVA). Asterisks denote significant difference (P<0.05, P<0.01, P<0.01, P<0.001, P<0.01 and P<0.001 for liver 8°C 48 h, gills 26°C 6 h, gills 26°C 24 h, gills 26°C 48 h, gills 8°C 6 h and kidney 8°C 48 h, respectively) to normoxic fish at the same temperature (Holm–Sidak post test).
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