Image processing software

HEK293-AT1 cells were transfected with the NES-GFP-PKDC1ab (W166A) or NES-dsRed-NES-Spo20m construct. After 24 hr, cells were imaged in a Zeiss LSM 780 confocal microscope at room temperature. After stimulation with 100 nM AngII, translocation of the fluorescent probe was monitored in time-lapse imaging. The translocation of the construct from the membrane to the cytosol was quantified by measuring cytosolic fluorescent intensity in regions of interest outside the nucleus and plotted against time using the Zeiss image-processing software. Intensity curves were normalized to pre-stimulatory values, and they were averaged from the indicated number of cells in recordings obtained in several dishes in multiple independent experiments.

To quantify Lectin re-perfusion into the infarct area, confocal images taken with 10X objective of Lectin perfused P2 or P7 injured hearts were analyzed 4 days post-MI. Using Zeiss image processing software, mean fluorescence intensity (MFI) of Lectin was calculated from 4 non-infarct healthy regions and 4 infarct areas below the stitch for each heart injured at P2 or P7. MFI was measured from equivalent areas across all regions and hearts. For each heart, the 4 MFI measurements obtained from mal-perfused infarct areas below the stitch were plotted relative to average of MFIs obtained from healthy well-perfused areas for that same heart.