Figure 5.

AID knockdown in human BM cells causes skewed differentiation toward myelomonocytic lineage. (A) Schema of short hairpin RNA (shRNA) construct against human AID cloned into pLKO.1 vector. (B) Human BM CD34⁺ cells were transduced with 3 lentiviral vectors expressing short hairpins (sh) against human AID (hAID), and mRNA expression was measured by RT-PCR. Data were analyzed by the Δ Ct ratio technique using human HPRT gene as a housekeeping gene. Results are presented as the ratio of the sh-AID value to the scramble value. The data are mean ± SD (n = 3 for each hairpin). (C) Scramble or sh-3 transduced human BM CD34⁺ cells were cultured in serum-free StemSpan medium supplemented with IL-3, FLT3L, stem cell factor (SCF), and granulocyte colony-stimulating factor (G-CSF) to monitor GM differentiation in vitro. The left figure shows the representative immunophenotype of CD14/CD15 in cultured cells. The right bar graph shows the percentage of CD14⁺/CD15⁻ or CD14⁻/CD15⁺ cells in cultured cells. The data are mean ± SD (n = 3 for each arm). (D) Scramble or sh-1 transduced human BM CD34⁺ cells were cultured in methylcellulose medium (H4434) for 9 days and colony numbers were counted. Left bar graph shows the number of GM and CFU-E colonies in each arm. Right bar graph shows the percentage of GM and CFU-E colony in each arm. Thirty thousand cells were plated per well in triplicate. Data are representative of 2 independent experiments. The data are mean ± SD. *P = .0172 (percentage of CFU-E in Scr vs AID-KD). (E) Representative images of CFU-E colony derived from scramble or AID-silenced (sh-1 transduced) cells in indicated magnification. 5′LTR, 5′ long terminal repeat; cPPT, central polypurine tract; hPGK, human phosphoglycerate kinase; KD, knockdown; PuroR, puromycin resistance gene; RRE, Rev response element; Scr, scramble; Sin 3′LTR, self-inactivating 3′ long terminal repeat; U6, human U6 promoter.