Fig. S6

The effect of inhibition, knockdown and knockout of Hsp90 and the Tor pathway on PGC number and migration. (A) Images of embryos at 24 hpf following treatment with low levels of the different inhibitors (17-DMAG (35 µM), rapamycin (50 µM)), with ectopic PGCs indicated by arrowheads and the migration target with an asterisk. The PGCs express farnesylated EGFP on their membrane. (B) Scatter plot presenting the percentage of PGCs located in ectopic positions following treatment of embryos with low levels of the indicated inhibitors. Results derived from three experiments. All treatments were analyzed using Mann-Whitney-test to compare with DMSO (control), resulting in the following p-values: to rapamycin: p=0.1232; to 17-DMAG: p=0.1729; to 17-DMAG and rapamycin combined: p <0.0001. N is the number of embryos analyzed. (C) The average number of PGCs in 24 hpf embryos treated with either DMSO or 50 µM 17-DMAG. The results are derived from three experiments, the p-value was calculated using t-test with p=0.3802. N is the number of embryos analyzed. n.s.=not significant, error bars indicate SEM (D) The average number of PGCs per embryo at 24 hpf is not affected by manipulating the level of the Hsp90aa1.2 protein. The data is derived from three experiments and statistical analysis with t-test shows no significant difference; control to knockdown: p=0.0872; control to rescue: p=0.0879; knockdown to rescue: p=0.9238. N is the number of embryos analyzed. (E) The number of PGCs is not affected by the lack of Hsp90aa1.2 in mutant embryos as compared with mutant embryos supplemented with wild-type hsp90aa1.2. Mutant alleles used in this experiment were four times the hsp90aa1.2d34, one time the hsp90aa1.2d5 and one time the hsp90aa1.2in1. Significance level calculated using t-test: p=0.5790. N is the number of embryos analyzed.