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Fig. 3 | BMC Biology

Fig. 3

From: The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra

Fig. 3

Functional analysis of HAS-7 knockdown. a Detection of HAS-7 protein in body lysate samples using a polyclonal HAS-7 antibody. M = protein marker. Tubulin was used as loading control of the respective hydra lysates. The arrow indicates the position of the HAS-7 protein band, also in b and c. b Recombinant HAS-7 protein expressed in High Five cells compared to native protein in HL as detected by a HAS-7-specific antibody. Note that the slightly higher apparent molecular mass is due to the introduced histidine tag of recombinant HAS-7. c Knockdown effect of different siRNA combinations on HAS-7 protein levels was assayed by anti-HAS-7 Western blot analysis of complete hydras treated as indicated. Tubulin was used as loading control of the respective hydra lysates. The distinct band at 70 k Da in a-c likely represents a dimer of processed HAS-7. d HyWnt3-His proteolysis is impaired in HL of animals electroporated with siHAS-7 (2 + 3) as compared to siGFP control animals. Head lysates were generated 6 days after electroporation. Tubulin was used as loading control of the respective HL applied for each time point. e Relative intensities of the Western blot bands in d. f Quantitative real-time PCR analysis of HAS-7 expression in head tissues confirms the decreased expression in siHAS-7 treated animals compared to siGFP treated and untreated (steady-state AEP animals) controls. Relative expression levels are given in 2(-ΔΔCt). Results represent mean +/− S.D. from 3 independent experiments, analyzed by t tests. *p < 0.05. The individual data values are shown in Additional file 14. g siGFP control electroporation without AZK treatment shows normal morphology. Scale bar = 500 μm. h siHAS-7 electroporation without AZK treatment shows animals with double axis lacking ectopic tentacles. Scale bar = 200 μm. i siGFP control animal showing ectopic tentacle formation after AZK treatment. Scale bar = 200 μm. j Double axis phenotype with ectopic tentacles near the head region in hydras treated with AZK after HAS-7 (2 + 3)/GFP siRNA electroporation. The asterisk denotes the peduncle region. Scale bar = 500 μm. k Both heads in HyWnt3P::HyWnt3 transgenic animal treated as in j exhibit hypostomal HyWnt3 expression (arrow). Smaller spots along the body column represent ectopic organizers that usually give rise to ectopic tentacles as in i. Scale bar = 200 μm. l Ectopic axis phenotype in actin::HyWnt3 transgenic hydra. Red arrows indicate multiple secondary heads. m No double axis was observed in hydras after HyDkk1/2/4/GFP siRNA electroporation. Scale bar = 500 μm. n Double axis phenotype in hydras after HyDkk1/2/4/GFP siRNA electroporation and AZK treatment. Scale bar = 500 μm. o Rescue of double axis phenotype in animals treated with AZK after electroporation with a combination of HAS-7 (2 + 3) and HyWnt3 siRNAs. Scale bar = 200 μm. p No double axes were observed in hydras treated with AZK after electroporation with HAS-1/GFP siRNAs. q Ratios of double axis phenotypes in hydras after electroporation with siGFP or combinations of siRNAs as indicated. In animals without subsequent AZK treatment double axes were counted 6 days after electroporation. In animals treated additionally with AZK, incubation was started 6 days after electroporation and the numbers of double axes in each group were counted 5 days after AZK removal. Total numbers of animals with double axis phenotype in each group were: siGFP/DMSO = 0/192 (n=5), siGFP/AZK = 10/230 (n=5), siHAS-7/siGFP = 90/186 (n=4), siHAS-7/siGFP/AZK = 203/248 (n=6), siHAS-7/siHyWnt3/AZK = 11/203 (n=3), siHyDkk1/2/4/siGFP/AZK = 65/290 (n=3), siHMP1/siGFP/AZK = 93/204 (n=3), siHAS-1/siGFP/AZK = 1/150 (n=3). Results from at least three independent experiments are shown. Each column represents the total percentage of one group, bars indicate the mean ± S.E.M. ****P value < 0.0001, ***P value < 0.0005, **P value < 0.001. ns = not significant. The data were analyzed using an unpaired parametric T test with Welch’s correction followed by pairwise multiple comparisons of each group with the other groups. The individual data values are shown in Additional file 14

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