Eif6 accumulation in patients with SDS and sbds-KO zebrafish.

Western blots showing EIF6 accumulation in SDS lymphoblasts (A), SDS PBMCs (B), and sbds-KO fish from 5 dpf to 10 dpf (C). CTL1, healthy control no. 1. (D) IHC images of Eif6 expression showing accumulation in the liver and digestive tract (DT) of 10 dpf zebrafish. Original magnification, ×40.

Eif6 is essential for zebrafish embryonic development.

(A) Amino acid alignment of WT and eif6 KO zebrafish; the insertion of 1 bp causes early truncation. (B) mRNA eif6 levels by RT-qPCR on the tp53^+/+ and tp53^M214K/M214K backgrounds at 5 dpf. (C) Western blots showing the absence of Eif6 protein in the eif6^–/– zebrafish larvae at 5 dpf. Note the dose effect in the eif6^+/– fish compared with their WT siblings. (D) Western blot quantification. (E) eif6 KO showed a significantly lower number of neutrophils than the WT siblings at 5 dpf. (F) Sudan black staining for neutrophils counts (original magnification, ×2.5 and ×8). (G) Survival percentages for eif6^+/+, eif6^+/–, and eif6^–/– siblings at 5, 8, and 10 dpf. *P < 0.05, **P < 0.01, and ***P < 0.001, by ANOVA. Data represent the mean ± SEM.

Low levels of Eif6 are enough for survival to adulthood, but only an absence of eif6 KO affects the levels of ribosomal proteins.

(A) Amino acid alignment of the 2 eif6 mutants (eif6^ms/ms and eif6^{del2aa/del2aa}) compared with the Eif6 WT sequence. (B) mRNA levels in all 3 eif6 mutants and WT zebrafish at 5 dpf. (C) Western blots showing Eif6, Sbds, and RP levels in the different eif6 mutants. (D) Western blot quantification. (E) Western blots showing Rpl23 and Rps3 in eif6^+/+, eif6^+/–, and eif6^–/– siblings at 5 dpf. (F) Western blot quantification relative to actin. (G) Western blotting showing Rpl23 and Rps3 expression in eif6^+/+, eif6^{del2aa/del2aa}, and eif6^ms/ms zebrafish at 5 dpf. (H) Western blot quantification relative to actin. (I) Polysome profiles of eif6 WT and mutants at 5 dpf. *P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA. Data represent the mean ± SEM.

Tp53 pathway activation in eif6 mutants does not affect survival rates.

RT-qPCR analysis of tp53 and its targets of WT and eif6 KO on the (A) tp53^+/+ and (B) tp53^M214K/M214K backgrounds. (C) Scheme of the double-heterozygote crosses (eif6^+/–tp53^+/M214K). (D) Percentage of survival for double-heterozygote crosses of eif6^+/–tp53^+/M214K at 8 and 15 dpf. (E) RT-qPCR analysis of tp53 and its targets (*P < 0.05, **P < 0.01, and ***P < 0.001, by 1-way ANOVA), and (F) UPR markers in WT and eif6^ms/ms zebrafish at 5 dpf (*P < 0.05 and ***P < 0.001, by 2-tailed t test). Data represent the mean ± SEM.

Partial rescue of sbds KO with 1 copy of the WT eif6 allele.

(A) Scheme showing the incrossing of 2 double-heterozygotes (eif6^+/– sbds^+/–). (B) Genotype percentages at 15 dpf of an incrossing of double-heterozygotes. (C) Western blots showing Eif6 accumulation in sbds KO fish with only 1 copy of the eif6 WT allele (eif6^+/–). (D) Western blot quantification of Eif6 protein expression. (E) Sudan black staining to detect the number of neutrophils in 10 dpf siblings. Scale bars: 200 μm and 50 μm. Data represent the mean ± SEM.

Correlation of Eif6 levels with tp53 activation in sbds-KO zebrafish.

(A) Scheme of sbds^+/– crossings in the WT and Eif6 missense mutants and percentage of genotypes observed at 20 dpf. (B) Schemes of the 3 different crosses to obtain sbds WT and KO zebrafish on the eif6^+/+, eif6^+/ms, and eif6^ms/ms backgrounds. (C) Immunoblot showing Eif6 accumulation in sbds-KO zebrafish. (D) Neutrophil counts at 10 dpf. *P < 0.05, **P < 0.01, ***P < 0.001, by 1-way ANOVA with Dunnett’s test. (E) Protein levels in sbds^+/+, sbds^+/–, and sbds^–/–, in the eif6^+/+ and eif6ms/ms mutant backgrounds, along with protein quantification. (F) RT-qPCR results for sbds and eif6 (G) and the tp53 pathway. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s test. Data represent the mean ± SEM.

Tp53 activation in SDS patient–derived cells.

Western blots and quantification of (A) LCLs and (B) BM-MNCs. EIF6 mRNA (C) and protein (D) levels in 3 different siRNAs. (E) EIF6 mRNA levels in patients with SDS with and without siRNA (F) TP53 and CDKN1a mRNA levels are decreased after siRNA in patients with SDS. (G) Western blots showing decrease in TP53 levels after EIF6 knockdown using 3 different siRNAs. (H) TP53 protein quantification. Data represent the mean ± SEM. HD, healthy donor; NC, negative control.