Figure S4.
Effects of lamins on cluster polarity and protrusion dynamics. (A–B′) Schematics of polarity orientation (A and B) and merged images of control (A′) and LamRNAi (B′) clusters marked with E-cadherin to mark the apical surfaces, Dlg to mark basolateral surfaces, and Hoecsht to mark DNA. (A″–B‴) Dlg grayscale image. (A‴ and B‴) E-cadherin grayscale image. White arrowhead: polar cell apical cap; magenta arrowheads: lateral Dlg. (C–D′) Schematic (C and D) and inverted grayscale images (C′ and D′) of clusters stained for aPKC as an apical marker. Black arrowhead: polar cell apical cap; green arrowheads: apical border cell surface. (E and F) Images of border cells stained with Phalloidin to mark F-actin and Hoechst to mark DNA. (E′ and F′) Grayscale images of F-actin. (G) Schematic of line scale acquired across cluster. The binning of regions were for front: 0–10% length, middle: 11–89% length, and back: 90–100% length. (H) Individual and mean ± SEM F-actin levels in indicated clusters and position. Statistical test: Brown Forsythe and Welch followed by Dunnett’s Multiple Comparison; means for front, back, or middle F-actin are all not significantly different from LamRNAis for relative position, P > 0.9. (I) Individual and mean ± SEM ratios of the front:back intensity for each cluster. Statistical test: Kruskal–Wallis test, wRNAi versus Lam1RNAi: P > 0.9; wRNAi versus Lam2RNAi: P = 0.24. Number of clusters analyzed (n) in H and I: n = 78 (w), n = 134 (Lam1), and n = 95 (Lam2). (J) Schematic showing measurement of width in the leading cell. (K–N) Example plots showing the width of the protrusion tip, base, and nucleus for the indicated conditions for an individual cluster over time. (O) Plot with dots of individual and mean ± SEM correlations between nuclear width and base width for each cluster’s first protrusion. A Kruskal–Wallis Test was performed. N = 16 (wRNAi), N = 8 (Lam1RNAi), and N = 10 (Lam2RNAi) movies for each condition shown in C–E and J. Scale bars: 10 μm. Genotypes and experimental replicates reported in Table S2.

Effects of lamins on cluster polarity and protrusion dynamics. (A–B′) Schematics of polarity orientation (A and B) and merged images of control (A′) and LamRNAi (B′) clusters marked with E-cadherin to mark the apical surfaces, Dlg to mark basolateral surfaces, and Hoecsht to mark DNA. (A″–B‴) Dlg grayscale image. (A‴ and B‴) E-cadherin grayscale image. White arrowhead: polar cell apical cap; magenta arrowheads: lateral Dlg. (C–D′) Schematic (C and D) and inverted grayscale images (C′ and D′) of clusters stained for aPKC as an apical marker. Black arrowhead: polar cell apical cap; green arrowheads: apical border cell surface. (E and F) Images of border cells stained with Phalloidin to mark F-actin and Hoechst to mark DNA. (E′ and F′) Grayscale images of F-actin. (G) Schematic of line scale acquired across cluster. The binning of regions were for front: 0–10% length, middle: 11–89% length, and back: 90–100% length. (H) Individual and mean ± SEM F-actin levels in indicated clusters and position. Statistical test: Brown Forsythe and Welch followed by Dunnett’s Multiple Comparison; means for front, back, or middle F-actin are all not significantly different from LamRNAis for relative position, P > 0.9. (I) Individual and mean ± SEM ratios of the front:back intensity for each cluster. Statistical test: Kruskal–Wallis test, wRNAi versus Lam1RNAi: P > 0.9; wRNAi versus Lam2RNAi: P = 0.24. Number of clusters analyzed (n) in H and I: n = 78 (w), n = 134 (Lam1), and n = 95 (Lam2). (J) Schematic showing measurement of width in the leading cell. (K–N) Example plots showing the width of the protrusion tip, base, and nucleus for the indicated conditions for an individual cluster over time. (O) Plot with dots of individual and mean ± SEM correlations between nuclear width and base width for each cluster’s first protrusion. A Kruskal–Wallis Test was performed. N = 16 (wRNAi), N = 8 (Lam1RNAi), and N = 10 (Lam2RNAi) movies for each condition shown in C–E and J. Scale bars: 10 μm. Genotypes and experimental replicates reported in Table S2.

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