Knockdown of Adpgk in embryonic zebrafish results in aberrant body axis development and cell death. (a) RT-qPCR analysis of adpgk and hexokinase 1 (hk1) expression in wild type embryos collected at different developmental stages. N = 4 independent experiments. (b) Immunoblotting of Adpgk after blocking correct splicing (KD1) or translation (KD2) of adpgk mRNA by morpholino (MO) technology in zebrafish embryos, compared to wild type and control MO treated animals. Representative images, N = 3 independent experiments. (c) Representative images of aberrant body axis development in KD1 zebrafish ranging from shortened body to strong dorsalization (stage 60 hpf; lateral views with anterior to the left). Hypomorphic zebrafish are compared to age matched wild type and control MO treated animals. (d) Representative images of TUNEL staining in wild type and KD1 embryos (stage 22 hpf; lateral views with anterior to the left). (e) RT-qPCR analysis of bcl, bbc3 and bcl2l expression in wild type and KD1 embryos as wells as KD1 embryos rescued with adpgk mRNA (stage 8 hpf). N = 5 independent experiments. (f) RT-qPCR analysis of the cell cycle checkpoint genes cdkn1a and gadd45aa in wild type and KD1 embryos as well as KD1 embryos rescued with adpgk mRNA (stage 8 hpf). N = 5 independent experiments. (i) RT-qPCR analysis of CDKN1A expression in control and ADPGK-deficient Jurkat T cells after different periods of stimulation with and without PMA. N = 3 independent experiments. * p < 0.05, ** p < 0.01, ***p < 0.005. All images of blots represent cropped blots of appropriate protein size. For full length blots see Supplemental Fig. 3.
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