Fig. S3

Implication of calcium signaling from the ER in nrz morphants phenotype

(A)Thapsigargin injected embryos (5μM) showing margin constriction and blastomere detachment (black arrowheads, right panel), as compared to DMSO-injected controls (left panel). (B) Mitotracker™ labeling of YSL mitochondria showing the dramatic decrease of the ΔΨ_m of thapsigargin-treated embryos, compared to control embryos. (C) Immunoblot analyses. Detection of cytochrome C in YSL mitochondria isolated from embryos treated with increasing doses of Thapsigargin (μM) (bottom panel). The same western blot was probed with anti-Nrz (middle panel) and anti-F0/F1 ATPase (top panel, loading control) antibodies. (D) Histogram showing the effect of 2-APB treatement on epiboly progression (n : number of injected embryos); control embryos were treated with DMSO . (E) False color images of zebrafish embryos injected at 1-cell stage with nrzMO and Oregon Green BAPTA-1 AM, and re-injected at 512-cells stage with MOCK, heparin (400 g/mL) or Xestospongin C (20μM). (F) Embryo mortality measured at 9hpf. Number of injected embryos is shown (n). (G) Representative response of Fura-2 loaded HeLa cells either transiently expressing Nrz, NrzCytB5, ΔBH4NrzCytB5 or transfected with empty vector (pCS2+). Cells were stimulated with 5 μM histamine, an IP3-generating agonist (Peppiatt et al., 2003) during the indicated time (horizontal bar), and the kinetics of the subsequently Ca2+ release was measured by fluorescence as described in supplementary materials and methods. Representative data from 3 independent experiments. (H) Quantification of the histamine-induced Ca2+ peak. Means of 3 independent experiments, error bars are shown.