Table 1.
FACS antibodies used in this study.
Fig 1.
RIOK2 is essential for the proliferation of mature blood cells in vivo and for mouse survival.
A) Schematic overview of the competitive bone marrow transplantation. Lethally irradiated B6/SJL mice were injected with a mix of bone marrow cells from Riok2fl/fl; Rosa26::CreERT2 or Riok2fl/+; Rosa26::CreERT2 mice and cells from wild-type B6-SJL mice in a 1:1 ratio. 4 weeks after transplantation, all animals were injected intraperitoneally with tamoxifen to induce Cre-mediated recombination of the Riok2 locus. Created with Biorender.com. B) CD45.2 chimerism in the indicated peripheral blood cell types 4, 8, 12 and 16 weeks after bone marrow transplantation. Data is represented as mean ± standard deviation (SD). n = 4 animals per group. **: p<0.01; ***: p<0.001 by Unpaired t-test. C) Bar graphs depicting the absolute number of CD45.2 positive cells in each indicated population of mice transplanted with Riok2fl/fl; Rosa26::CreERT2 treated with corn oil (control) or tamoxifen. Data is represented as mean ± standard deviation (SD). n = 4 animals per group. **: p<0.01; ****: p<0.0001 by 2way ANOVA test followed by Tukey’s multiple comparison test. D) Kaplan-Meier survival curves of the indicated transgenic mouse strains after deletion of Riok2 using tamoxifen (left panel) or polyIC (right panel). n = 4 animals per group. E) Bone marrow histology revealed by HE-staining of bone marrow from Riok2+/+, Riok2fl/+, or Riok2fl/fl; Rosa26::CreERT2 mice collected 20 days after tamoxifen treatment or upon death. Scale bar represents 100 μm.
Table 2.
Sequences for PCR primers used in this study.
Table 3.
Western Blot antibodies used in this study.
Fig 2.
RIOK2 loss affects differentiation and proliferation of hematopoietic stem and progenitor cells in vitro.
A) Experimental outline to determine the effects of RIOK2 loss on hematopoietic stem and progenitor cells in vitro. Created with Biorender.com. B) Percentage of Lin- cells and LSK cells 48h after the addition of either ethanol (EtOH) or 4-hydroxytamoxifen (OHT) to the culture medium of Riok2fl/fl; Rosa26::CreERT2 and Riok2fl/+; Rosa26::CreERT2 cells. Data is represented as mean ± standard deviation (SD). n = 3 biological replicates per group. A Student’s t-test was performed to assess statistical significance (ns = not significant, *p<0.05, ***p<0.001). C) MethoCult replating assay using LSK cells sorted from bone marrow of the indicated genotypes. Cells were replated once per week. Data is represented as mean ± standard deviation (SD). n = 3 biological replicates per group. D) Left panel: EdU labeling of Riok2fl/fl; Rosa26::CreERT2 and Riok2fl/+; Rosa26::CreERT2 LSK cells. Error bars represent standard deviation (SD), n = 3 biological replicates. Right panel: representative FACS plot of Riok2fl/fl treated with either EtOH or OHT for 5 days.