Figure 3 FOXP1 promotes expansion of... | American Society of Hematology

Figure 3

$Figure 3. FOXP1 promotes expansion of primary human B cells, not by stimulating proliferation but by repressing apoptosis. Memory B cells were sorted from human peripheral blood and transduced with FOXP1-IRES-YFP, BCL6-IRES-GFP, or ctrl-IRES-YFP and cultured with CD40L-L cells, IL-21, and IL-2. (A) Representative example of FOXP1 and BCL6 overexpression in primary YFP+ or GFP+ B cells. Six days after transduction, YFP+ cells were sorted and analyzed by immunoblotting. β-Actin was used as loading control. (B) FOXP1-IRES-YFP, BCL6-IRES-GFP, and ctrl-IRES-YFP transduced B cells were continuously cultured with IL-21 and IL-2 and CD40L-L cells. The percentage of transduced cells in each culture was followed over time by FACS analysis and normalized to the percentage of transduced cells at day 3 after transduction. Mean ± standard deviation (SD) of 3 independent experiments are shown. (C) Seven days after transduction, cells were labeled with eFluor 670 and cultured for 4 days, after which the eFluor 670 intensity was determined by flow cytometry. Representative graphs of 3 independent experiments are shown. (D) A total of 6 to 7 days after transduction, live YFP-positive and YFP-negative fractions of FOXP1 and control vector single-transduced cultures were sorted. After a recovery period of 4 to 5 days, the percentage of cells in the forward-scatter/side-scatter live gate was determined by flow cytometry at 3 consecutive time points. The data were normalized to the percentage of living cells measured at the first time point. Mean ± standard error or the mean (SEM) of 2 independent experiments are shown. (E) A total of 6 to 7 days after transduction, the YFP-positive fractions of FOXP1- and control vector–transduced cultures were sorted. After a recovery period of 5 to 7 days, caspase-3/7 activity was determined by the Caspase-Glo 3/7 assay. Values were corrected for number of living cells as determined by FACS analysis. Mean ± SD of 4 independent experiments are shown (Student t test, *P < .05). (F-G) DLBCL cell lines were nucleofected with control siRNA or siRNA against FOXP1. (F) Four days after nucleofection, caspase-3/7 activity was determined by the Caspase-Glo 3/7 assay. Values were corrected for number of living cells as determined by FACS analysis. Mean ± SEM of 2 independent experiments are shown. (G) Cell lines were labeled with CFSE 1 day before nucleofection. Three days after nucleofection, CFSE intensity was determined by flow cytometry. Representative graphs of 2 independent experiments are shown. Efficient knockdown in these experiments was validated by qRT-PCR analysis.$

FOXP1 promotes expansion of primary human B cells, not by stimulating proliferation but by repressing apoptosis. Memory B cells were sorted from human peripheral blood and transduced with FOXP1-IRES-YFP, BCL6-IRES-GFP, or ctrl-IRES-YFP and cultured with CD40L-L cells, IL-21, and IL-2. (A) Representative example of FOXP1 and BCL6 overexpression in primary YFP⁺ or GFP⁺ B cells. Six days after transduction, YFP⁺ cells were sorted and analyzed by immunoblotting. β-Actin was used as loading control. (B) FOXP1-IRES-YFP, BCL6-IRES-GFP, and ctrl-IRES-YFP transduced B cells were continuously cultured with IL-21 and IL-2 and CD40L-L cells. The percentage of transduced cells in each culture was followed over time by FACS analysis and normalized to the percentage of transduced cells at day 3 after transduction. Mean ± standard deviation (SD) of 3 independent experiments are shown. (C) Seven days after transduction, cells were labeled with eFluor 670 and cultured for 4 days, after which the eFluor 670 intensity was determined by flow cytometry. Representative graphs of 3 independent experiments are shown. (D) A total of 6 to 7 days after transduction, live YFP-positive and YFP-negative fractions of FOXP1 and control vector single-transduced cultures were sorted. After a recovery period of 4 to 5 days, the percentage of cells in the forward-scatter/side-scatter live gate was determined by flow cytometry at 3 consecutive time points. The data were normalized to the percentage of living cells measured at the first time point. Mean ± standard error or the mean (SEM) of 2 independent experiments are shown. (E) A total of 6 to 7 days after transduction, the YFP-positive fractions of FOXP1- and control vector–transduced cultures were sorted. After a recovery period of 5 to 7 days, caspase-3/7 activity was determined by the Caspase-Glo 3/7 assay. Values were corrected for number of living cells as determined by FACS analysis. Mean ± SD of 4 independent experiments are shown (Student t test, *P < .05). (F-G) DLBCL cell lines were nucleofected with control siRNA or siRNA against FOXP1. (F) Four days after nucleofection, caspase-3/7 activity was determined by the Caspase-Glo 3/7 assay. Values were corrected for number of living cells as determined by FACS analysis. Mean ± SEM of 2 independent experiments are shown. (G) Cell lines were labeled with CFSE 1 day before nucleofection. Three days after nucleofection, CFSE intensity was determined by flow cytometry. Representative graphs of 2 independent experiments are shown. Efficient knockdown in these experiments was validated by qRT-PCR analysis.

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