Aspects Affecting the Expression of Hypoxia-Inducible Factors in Cardiomyocytes



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By comparing the immunofluorescent images of each of the wells containing cells in different conditions, we can compare signals that are relevant to diabetic cardiomyopathy change HIF expression, activity, or localization. The cells were put in normal media, CoCl₂, or isoproterenol, and were transfected with HA HIF-1α and HA HIF-2α. We added only the 2nd antibody to one of the wells for a negative control. The signals from this well would show how much of the other signals should be expected as background. We knew from prior research that CoCl₂ should increase HIF expression and used cells in CoCl₂ as a positive control. The mouse HIF-1α antibody (ab113642) can be detected using cyanine 5 dye (CY5) because it emits a wavelength of 488. Similarly, the rabbit HIF-2α antibody (PA1-16510) can be detected using green fluorescent protein (GFP) due to its wavelength of 647. The results supported previous studies from other cell types. The cells in CoCl₂ were transfected with HIF-1α and HIF-2α overexpressed the HIFs in comparison to those in normal media. Additionally, we found that cells treated to induce hypertrophy also may increase HIF expression.