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. 2020 Nov 16;11:5816. doi: 10.1038/s41467-020-19658-0

Fig. 3. Mutation of RRP7A inhibits neurogenesis.

Fig. 3

a WB analysis of RRP7A expression in CRISPR clones of P19CL6 mouse stem cells holding WT RRP7A (P19CL6Rrp7aWT#1 and P19CL6Rrp7aWT#2) or mutated RRP7A; P19CL6Rrp7aΔ1/Δ18 (pArg59LysfsTer60/p.53-58del) and P19CL6Rrp7aΔ8/Δ33 (p.Arg48ProfsTer15/p.49-59del). b IFM analysis of CRISPR clone proliferation at day 3 post seeding in the absence of retinoic acid (RA). Nuclei were stained with DAPI (blue). Dotted lines outline cell colonies. Scale bars, 1 mm. c Quantification of proliferation of CRISPR clones measured by cell coverage (confluency) (data average of n = 3 independent experiments); Rrp7aWT#2 (P = 0.230), Rrp7aΔ1/Δ18 (P = 1.268E-05), Rrp7aΔ8/Δ33 (P = 5.372E-04)). d Quantification of relative levels of BrdU incorporation in WT and mutant clones (data average of n = 3 independent experiments; Rrp7aWT#2 (P = 0.617), Rrp7aΔ1/Δ18 (P = 0.004), Rrp7aΔ8/Δ33 (P = 5.635E-04)). e, f IFM analysis of the ability of CRISPR clones to undergo neurogenesis after 6 d and 9 e days of RA stimulation. The stippled lines in the upper panels indicate the lining of cellular clusters from where neurons are formed in WT clones. Neurons are marked with anti-ßIII-tubulin (upper panels: white; lower panels: green) and anti-MAP2 (lower panels: red), and nuclei are stained with DAPI (blue). Lower panels are shown as shifted overlays between ßIII-tubulin and MAP2. Scale bar upper panels, 0.1 mm and lower panels, 40 µm. g Quantification of relative levels of βIII-tubulin fluorescence in WT and mutant clones shown in f (data average of n = 3 independent experiments; Rrp7aΔ1/Δ18 (P = 6.244E-05), Rrp7aΔ8/Δ33 (P = 2.479E-04)). h WB analysis of WT clone P19CL6Rrp7aWT#1 and mutant clone P19CL6Rrp7aΔ8/Δ33 at day 3, 6, and 9 of RA stimulation; antibodies as indicated. i–k Quantification of relative levels of PAX6 (day 3: P = 0.532; day 6: P = 2.696E-05; day 9: P = 0.025) (i), ßIII-tubulin (day 3: P = 0.232; day 6: P = 0.003; day 9: P = 2.400E-05) (j) and MAP2 (day 3: P = 0.774; day 6: P = 0.085; day 9: P = 1.117E-07) k shown in h (data average of n = 3 independent experiments). Data are represented as mean ± SD and significance was determined using an unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.: not significant.