|
Fig. 5
Genetic repression of miR-29 chronically affects iron homeostasis. a, b Western blot of IRP2 and TFR1A in 12-week-old tg(kif5aa:eGFP-sponge-29) (n?=?4) and wild-type (n?=?4) fish brain extracts and (b) relative densitometric analysis (*P?<?0.05; **P?<?0.01, Mann?Whitney U-test). c Representative Western blot of IRP2 in brain extracts of kif5a:sponge-29 at different ages (5, 12, 20, 27). In a?c ?-TUBULIN was used as loading control. d Comparison of densitometric analysis calculated on Additional file 8a and b. The blue line represents IRP2 expression in tg(kif5aa:eGFP-sponge-29) animals, gray line represents expression in wild-type fish. Values were normalized to the mean of 5 weeks value (Kruskal?Wallis test, P?=?0.0939, n?=?3 animals for each age point). e, f Expression level at 24 hpf of Ireb2 and Tfr1a upon miR-29 mimics injection. The expression level was determined by RT-qPCR. Statistical significance was assessed by one-way ANOVA with post-hoc Tukey?s test (*P?<?0.05), the analysis was performed on total RNA extraction from 30?40 embryos for each condition. Error bars indicate standard errors of means. g Brain non-heme iron content (?g/g wet tissue) in kif5a:eGFP-sponge-29 (n?=?5) compared to wild-type (n?=?6) at age 12 weeks (*P?<?0.05, Mann?Whitney U-test). h Representative images of lipofuscin accumulation in the optic tectum of 12-week-old kif5a:eGFP-sponge-29 and wild-type fish brains. Lipofuscin auto-fluorescent granules (green) were detected with ApoTome microscope, counterstained with DAPI (blue). Scale bar: 50 ?m. i Quantification of lipofuscin density based on percentage of area over threshold, n?=?6 (*P?<?0.05; Mann?Whitney U-test)