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Supplementary Figure 5 Knock-down of Pgam5 did not cause increased cell death. (A) Embryos were injected with 0.4 pmol Control MO, Pgam5 MO1 or Pgam5 MO2 into both blastomeres at the two-cell stage. At NF stage 12.5 embryos were stained for dead cells with 1 μg/ml Acridine Orange in 0.1x MBSH for 30 min, washed three times with 0.1x MBSH and imaged using a Leica M165FC stereomicroscope equipped with epifluorescence optics and a Jenoptik ProgRes C14 camera. As positive control, embryos were treated at NF stage 11.5 for 2h with 1μM staurosporine or buffer only, stained with Acridine Orange and imaged as described above. Image acquisition parameters were kept constant for all images. Mean fluorescence intensities were measured for each embryo using the Image J software package. The images show representative embryos injected with Control MO, Pgam5 MO1 or Pgam5 MO2 as well as staurosporine-treated embryos. The average fluorescence intensities ± SD are plotted in the graph; values did not differ significantly between Control MO, MO1 and MO2 (separate variances t-test, p-values > 0.7), staurosporine treatment significantly increased Acridine Orange fluorescence intensity compared to control embryos (** p-value < 0.01). The total numbers of analyzed embryos from four independent experiments are given below each column. (B) Embryos were injected with 0.4 pmol Control MO or Pgam5 MO1 and co-injected with 100 pg pCS2 LacZ into one blastomere at the four-cell stage. As positive control, embryos were treated with 1μM staurosporine for 1h at NF stage 19. At NF stage 20 embryos were fixed and stained for β- Galactodidase. Cleaved Caspase 3 (Cleaved Casp3) was detected by whole-mount immunostaining and colorimetric Alkaline Phosphatase staining (NBT/BCIP). For MO-injected embryos Cleaved Casp3- positive cells on injected and uninjected sides were compared and categorized into equal (low/high) or increased numbers in the injected side. Staurosporine-treated embryos were compared with control embryos and categorized into low or high number of Cleaved Casp3-positive cells. The graph summarizes the results from four independent experiments (** p-value < 0.01 χ2-test). The total numbers of embryos are provided below each column. (C) Embryos were injected with 1.6 pmol Control MO or 0.4 pmol, 0.8 pmol or 1.6 pmol Pgam5 MO1 and cultured till NF stage 40. The graph shows the average survival rates from at least three independent experiments. The total numbers of embryos are provided below each column. Injection of 0.4 pmol MO1 did not significantly affect survival, whereas survival rates dropped significantly after injection of higher doses of MO1 (separate variances t-test, * p-value < 0.05).

Image published in: Rauschenberger V et al. (2017)

Copyright © 2017. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.

casp3.2.LCASP-3, Apopain, xCPP32, caspase 3, caspase-3X.laevisThroughout NF stage 21embryo

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