Fig. 3

PLC–PKC activity is essential for polarisation at the 8-cell stage. a Scheme of the inhibitor (sphingosine, calphostin C) treatment. (b) DMSO, sphingosine and calphostin C-treated embryos immunostained for F-actin and Pard6. c, d Cell-contact-free enrichment of F-actin (c) or Pard6 (d) as a function of the IEA in embryos from (b). Each dot represents an individual measurement. e Percentage of polarised embryos in the groups of (b). The number of embryos per category is indicated. Embryos that did not show any blastomere with the enrichment of F-actin and Pard6 to the cell-contact-free surface were considered non-polarised. Otherwise embryos were classified as polarised. Data are shown as a contingency table. ****p < 0.0001, Fisher’s exact test. f Quantification of polarisation in embryos from (b). Data are shown as mean ± s.d. ****p < 0.0001, unpaired two-tailed Student’s t test (three independent experiments). g Scheme of U73122 treatment and OAG rescue experiment. h DMSO, U73122 and U73122 + OAG-treated embryos immunostained for F-actin and Pard6. i, j Cell-contact-free enrichment of F-actin (i) or Pard6 (j) as a function of the IEA in embryos from panel h. Each dot represents an individual measurement. k Percentage of polarised embryos in the groups of (h). The number of embryos per category is indicated. Data are shown as a contingency table. ****p < 0.0001, NS = not significantly different, Fisher’s exact test. l Quantification of polarisation in embryos from (h). Data are shown as mean ± s.d. ***p < 0.001, ****p < 0.0001, NS = not significant different, Kruskal–Wallis test and Dunn’s multiple comparisons test (two independent experiments). m Scheme of the PLC-DN overexpression and OAG rescue experiment. n Ruby and PLC-DN overexpressing embryos ± OAG immunostained for F-actin and Pard6. o Percentage of polarised embryos in the groups of (n). The number of embryos per category is indicated. Data are shown as a contingency table. ****p < 0.0001, **p < 0.01, Fisher’s exact test. p Quantification of polarisation in embryos from (n). Data are shown as mean ± s.d. **** p < 0.0001, **p < 0.01, *p < 0.05, Kruskal–Wallis and Dunn’s multiple comparisons tests (two independent experiments). All scale bars, 15 μm