Figure 6. Deletion of Rroid-E1 results in decreased mature NK cells and Id2 expression.

A. Schematic of locked nucleic acid (LNA) experiment. Sorted wild-type splenic NK cells were expanded ex vivo for 7d and electroporated with either 4μM control LNA or a 4μM mix of four LNAs targeting the Rroid RNA and cultured for either 6h or 24h.

B. qPCR analysis of Rroid (left) and Id2 (right) expression at the indicated time points following electroporation with LNAs (n=2 biological replicates). Data are representative of two independent experiments. Normalized to Hprt expression and shown as fold change relative to controls.

C. Gene browser ATAC-seq tracks showing open chromatin within the Rroid locus. Highlighted are open chromatin peaks and CRISPR/Cas9-targeted regions for the Rroid-P1 and Rroid-RE1 knockouts.

D. Chromatin conformation capture (3C) of interactions between the Id2 promoter and the indicated genomic regions. Data are normalized to interaction frequency of ligated BAC templates containing the regions of interest. Primer locations are indicated by arrows; the bait primer located in the Id2 promoter is indicated by the red arrow. (n=3 biological replicates). Data represent two independent experiments.

E. Gene browser view of STAT5 ChIP-seq showing binding at the Rroid locus in the presence or absence of IL-15 stimulation for 2h at 20ng/mL.

F. Fold change in Rroid expression in splenic NK cells sorted from 6–8 week old Rroid-P1^−/−, Rroid-RE1^−/−, and Rroid^+/+ littermate control mice as measured by qPCR (n=7 littermate controls (Rroid^+/+), 6 Rroid-P1^−/−, and 3 Rroid-RE1^−/− mice per group). Normalized to Hprt expression and shown as fold change relative to littermate controls.

G. Fold change in Id2 expression in splenic NK cells sorted from 6–8 week old Rroid-P1^−/−, Rroid-RE1^−/−, and Rroid^+/+ littermate control mice as measured by qPCR (n=7 littermate controls (Rroid^+/+), 6 Rroid-P1^−/−, and 3 Rroid-RE1^−/− mice per group). Normalized to Hprt expression and shown as fold change relative to littermate controls.

H. Absolute numbers of splenic NK cells in 6–8 week old Rroid-P1^−/−, Rroid-RE1^−/−, and Rroid^+/+ littermate control mice (n=9 Rroid^+/+, 6 Rroid-P1^−/−, and 3 Rroid-RE1^−/− mice per group).

I. Representative flow cytometry plots showing splenic NK cell maturation in in 6–8 week old Rroid-P1^−/−, Rroid-RE1^−/−, and Rroid^+/+ littermate control mice. Gated on live, CD45.2⁺ CD3, CD5, CD19⁻ NK1.1⁺ NKp46⁺ cells.

J. Frequency of maturing NK cell subsets. Splenic NK cells from 6–8 week old Rroid-P1^−/−, Rroid-RE1^−/−, and Rroid^+/+ littermate control mice were gated on live, CD45.2⁺ CD3, CD5, CD19⁻ NK1.1⁺ NKp46⁺ cells and analyzed for maturation markers (n=9 littermate controls (Rroid^+/+), 6 Rroid-P1^−/−, and 3 Rroid-E1^−/− mice per group).

*p≤0.05, **p≤0.01, ***p≤0.001. Two-tailed t-test. All error bars represent SEM. See also Figure S6.