Spe dm6 confocal microscope

Cells were stained for F‐actin fibers (post‐fixation in 4% paraformaldehyde [PFA]) using a conjugated phalloidin antibody (1:200, phalloidin‐AlexaFluor 488 [Invitrogen Carlsbad, California]) for 2 hours at room temperature. Cells were counterstained for nuclei visualization using 4′6‐diamidino‐2‐phenylindole (2 μg/mL; Sigma) for 10 minutes at room temperature. Slides were coverslipped and imaged on a Leica SPE DM6 confocal microscope (Wetzlar, Germany) using a 40× objective in oil immersion. Actin alignment was analyzed from images using the ImageJ plugin OrientationJ.⁵², ⁵³ OrientationJ characterizes the orientation and isotropic properties of a region of interest (ROI) of an image with actin visualized by labeling with phalloidin. ROIs were taken from the same geographically defined areas of each chamber well slide (totaling nine replicates each), to compute values or orientation and coherency. Coherency values specify the degree to which ROI features are oriented with a value of 1 indicating one dominant orientation and 0 if the local features are isotropic.⁵⁴, ⁵⁵ Differences in F‐actin coherency for NP cells cultured upon coated glass were determined using a one‐way ANOVA and Tukey's post hoc test.

Crosses for survival studies were set up in triplicates with 18–20 virgin females of KRAS TP53 PTEN APC crossed to 10–12 males at 29°C and parents were removed after 2 days of egg laying. KRAS TP53 PTEN APC virgins crossed to w¹¹¹⁸ males were used as baseline controls. The progeny were allowed to develop for the next 10 days at 29°C and survival to pupal stage was calculated by counting the number of experimental and control pupae as previously described(30 (link)). The size of the anterior hindgut area was measured using Image J(30 (link)). Cell number quantifications were performed on Leica SPE DM6 confocal microscope at 40X magnification. Statistical analysis was done using PRISM software.