Quantifying Latrunculin B's Effect on F-Actin

To quantify the effectiveness of latrunculin B in reducing F-actin levels [50 (link)], confluent HUVECs were serum starved for 4 h in 0.2% BSA medium and then treated with specified concentrations of latrunculin B for 30 min in 0.2% BSA medium. Subsequently, cells were treated with the same latrunculin B concentrations along with 1 pg/mL BMP9 for 45 min, fixed, and stained with phalloidin (see Immunofluorescence). For F-actin quantification, two Z-planes (2 µm interval) closest to apical and basal sides were analyzed for actin intensity, respectively. Three images from each condition for each experiment were captured using a spinning disk confocal microscope (as described above). Actin intensity was Z-projected by average intensity and quantified as the median across all fields using Fiji software (Version: 2.9.0/1.53t, U. S. National Institutes of Health, Bethesda, MD, USA).