FOXP1 represses PC differentiation of SKW6.4 and OCI-LY7 cells. (A-C) SKW6.4 and OCI-Ly7 cells were cultured without stimulation or were stimulated to differentiate with IL-21 and CD40L-L cells (OCI-Ly7) or IL-21 alone (SKW6.4) and were transduced with FOXP1-IRES-YFP or control empty vector. Four (OCI-Ly7) or 6 (SKW6.4) days after transduction, YFP⁺ cells were sorted. (A) Gene expression levels of FOXP1 as determined by qRT-PCR analysis in sorted cells. Expression levels were normalized to β-actin and then to expression levels in stimulated, control-transduced cells. Means ± SEM of 3 independent experiments are shown. (B) Equal numbers of sorted cells were cultured and analyzed by ELISA (left) or ELISPOT (right). Equal numbers of sorted cells (50 000) were cultured for an additional 24 hours. Thereafter, the supernatants were collected, and IgM protein levels were analyzed by ELISA. Levels were normalized to levels in stimulated, control-transduced cells. Means ± standard deviation (SD) of 5 (OCI-Ly7) or 4 independent experiments are shown. (left). Equal numbers of sorted cells were plated onto membranes in serial dilutions and cultured for an additional 18 hours, after which numbers of IgM-secreting cells were determined by ELISPOT. Spot numbers were normalized to numbers in stimulated, control-transduced cells. Means ± SD of 3 independent experiments are shown (right). (C) Gene expression levels of BCL6, PAX5, PRDM1, IRF4, and XBP1 in OCI-LY7 were analyzed by qRT-PCR. Expression levels were normalized to β-actin and then to expression levels in stimulated, control-transduced cells. Means ± SEM of 3 independent experiments are shown (1 sample t test, *P < .05; **P < .01; ***P < .001).