R221 treatment and IRAK4 knockdown differently affect the growth of xenografted human RS cells. (A) Treatment with R221 results in reduced growth of RS1316 and RS9737 cells in subcutaneous tumor and spleen but not in PC of xenografted NSG mice. Graphs show absolute number of leukemic cells at each site. Statistical analysis was done using the t test or Mann-Whitney test, as appropriate. (B) Immunoblotting analysis of IRAK4 expression in WT and IRAK4-edited RS9737 and RS1316 cells. (C) Indel analysis by amplicon capillary electrophoresis of the targeted region of IRAK4 in injected and recovered leukemia cells isolated from subcutaneous tumor, PC, and spleen. The WT allele is indicated by a red arrow, and mutant alleles are indicated by black arrows. (D) IRAK4 MAF in injected leukemia cells and cells isolated from subcutaneous tumors, PCs, and spleens of NSG mice. The tumor cells were recovered between 27 and 34 days after transplantation. Eleven independent experiments were performed, 3 each with RS1316, RS9737, and IP867/17 and 2 with RS1050 cells. Statistical analysis was done using 1-way repeated measures ANOVA.