For the analysis of Latrunculin B effects on F-actin, moss was cultured in PpNO3 media for 3–5 days, on top of cellophane disks. Pieces of cellophane containing protonemata were cut, flipped, and the protonemata were placed in direct contact with an agar pad containing Hoagland's medium and Latrunculin B at the indicated concentration. The cellophane was removed, 5 µl of liquid medium containing the same concentration of Latrunculin B were added, and a coverslip placed on top. The chamber was sealed with melted VALAP. Images were acquired with an interval of 10–20 min after chamber preparation. Control preparations contained DMSO at 0.2% in medium. Multiple cells and chambers were analyzed with identical results.
For the Latrunculin B sensitivity assay in moss, cells were prepared in the same way as for the growth assay (see above). Protoplasts were plated on small cellophane circles on top of agar in 96 well plates; wells were filled to the top with agar to create a flat surface to deposit the protoplasts. Cells were plated in protoplast regeneration medium in the absence of Latrunculin B for 4 days. At day 4 the cellophane discs were transferred to regular PpNH4 medium containing different amounts of Latrunculin B. Two days after transfer images were acquired from chlorophyll autofluorescence at a 30× zoom as 36-bit RGB color images with a CCD camera (Leica DF300FX) on a fluorescence stereo-microscope (Leica MZ16FA). Filter combinations were: excitation 480/40, dichroic 505 long pass, emission 510 long pass. The red channel of the color images, corresponding to chlorophyll fluorescence was digitally separated. The resulting 12-bit image was thresholded and the solidity estimated as mentioned above. Latrunculin treatments were performed in triplicate; a total of 7 to 38 plants was measured in each replicate. Dose response curves were fitted to the data using the sigmoidal fitting function of the program Origin (Microcal), using a logistic equation and a log10 scale for the concentration of Latrunculin B. The half maximal inhibitory concentration (IC50) was estimated from these fits. To compare the significance of the differences an ANOVA statistical test was used between the means obtained for each replicate. To calculate fractional solidity for each cell line and to plot the data, the following transformation was used: the minimum values obtained from the curve fitting were subtracted from the mean values; the resulting value was divided by the maximum value obtained by curve fitting.
For lily, bombarded pollen was grown on a slide and imaged according to our standard procedure (see above). The growth media was then replaced with fresh media plus 2 nM Latrunculin using a pipette. The procedure was performed twice to ensure that all of the media had been replaced.
For the Latrunculin B sensitivity assay in moss, cells were prepared in the same way as for the growth assay (see above). Protoplasts were plated on small cellophane circles on top of agar in 96 well plates; wells were filled to the top with agar to create a flat surface to deposit the protoplasts. Cells were plated in protoplast regeneration medium in the absence of Latrunculin B for 4 days. At day 4 the cellophane discs were transferred to regular PpNH4 medium containing different amounts of Latrunculin B. Two days after transfer images were acquired from chlorophyll autofluorescence at a 30× zoom as 36-bit RGB color images with a CCD camera (Leica DF300FX) on a fluorescence stereo-microscope (Leica MZ16FA). Filter combinations were: excitation 480/40, dichroic 505 long pass, emission 510 long pass. The red channel of the color images, corresponding to chlorophyll fluorescence was digitally separated. The resulting 12-bit image was thresholded and the solidity estimated as mentioned above. Latrunculin treatments were performed in triplicate; a total of 7 to 38 plants was measured in each replicate. Dose response curves were fitted to the data using the sigmoidal fitting function of the program Origin (Microcal), using a logistic equation and a log10 scale for the concentration of Latrunculin B. The half maximal inhibitory concentration (IC50) was estimated from these fits. To compare the significance of the differences an ANOVA statistical test was used between the means obtained for each replicate. To calculate fractional solidity for each cell line and to plot the data, the following transformation was used: the minimum values obtained from the curve fitting were subtracted from the mean values; the resulting value was divided by the maximum value obtained by curve fitting.
For lily, bombarded pollen was grown on a slide and imaged according to our standard procedure (see above). The growth media was then replaced with fresh media plus 2 nM Latrunculin using a pipette. The procedure was performed twice to ensure that all of the media had been replaced.
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