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Fig 8

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ZDB-IMAGE-201107-10
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Figures for Diofano et al., 2020
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Fig 8 Knockdown of <italic>upf1</italic> blocks mutant mRNA decay required for transcriptional adaptation in <italic>bag3<sup>-/-</sup></italic> mutants.

(A) Brightfield and birefringence images and densitometric analysis of birefringence (n = 4/6) of bag3+/+, bag3+/- and bag3-/- embryos at 72 hpf injected with 50μM MO-upf1. Individual samples are shown (One-way ANOVA followed by tukey's multiple comparison analysis, **P=0.0053, ***P=0.0002). (B)bag3-/- embryos injected with MO-upf1 develop (cardio-)myopathy (93.94±10.50%), whereas bag3+/+ (96.97±5.25%) and bag3+/- (91.09±8.82%) embryos are devoid of any phenotype (N = 3, n = 40, mean ± S.D., P<0.0001, two-tailed value for Fisher´s exact test). (C) Tropomyosin immunostainings of bag3+/+, bag3+/- and bag3-/- embryos injected with MO-upf1 at 72 hpf reveal muscle fiber disruptions due to the blockage of the transcriptional adaptation only in bag3-/- injected embryos. (D) Heart rate quantification at 72 hpf reveals impairments only in bag3-/- embryos injected with MO-upf1 (N = 3, n = 12, HR bag3+/+: 146±17.49 heart beat/min; HR bag3+/-: 130±19.18 heart beat/min; HR bag3-/-: 91±11.39 heart beat/min, mean ± S.D. One-way ANOVA followed by tukey's multiple comparison analysis, P<0.0001). (E) FS of ventricles of bag3-/- embryos injected with MO-upf1 is significantly reduced at 72 hpf (FS: 13.48±2.438%) compared to the FS measured in bag3+/+ (FS: 28.17±4.753%) and bag3+/- (FS: 25.23±4.328%) morphants (N = 3, n = 12; mean± SD One-way ANOVA followed by tukey's multiple comparison analysis, P<0.0001). (F)bag3-/- embryos injected with MO-upf1 (6.06±10.5%) reveal significant difference in responsiveness upon mechanical stimulus compared to bag3+/+(96.97±5.25%) and bag3+/- (91.09±8.82%) injected embryos (N = 3, n = 35, mean ± S.D. One-way ANOVA followed by tukey's multiple comparison analysis, P<0.0001). (G) Quantitative real-time PCR of bag3-/- +MO-upf1 and bag3-/- zebrafish embryos at 72 hpf shows restored bag3 mRNA levels only in bag3-/- embryos injected with MO-upf1 (N = 3, mean ± SD, P<0.0001 determined using two-tailed t-test). (H) Quantitative real-time PCR of bag3-/- embryos injected with MO-upf1and uninjected bag3-/- control embryos at 72 hpf reveals the downregulation of bag2 mRNA levels only in bag3-/- + MO-upf1 embryos (N = 3, mean ± SD, P = 0.0004 determined using two-tailed t-test).

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