Decreased ICOSL expression in EBNA2-transfected B-cell lymphomas. (Ai) Immunoblots showing EBNA2 expression in vector or EBNA2-transfected U2932 DLBCL. β-actin was used as a protein loading control. Densitometry analysis is shown in supplemental Figure 1B. (Aii) ICOSL MFI was assessed by flow cytometry. One representative experiment of 3 experiments showing a decrease in ICOSL in EBNA2-transfected U2932 cells. (Aiii) Histograms showing the average ICOSL MFI of 3 independent experiments by flow cytometry. (Bi) Immunoblots showing EBNA2 expression in the BJABK3 cell line transfected with a β-estradiol–inducible EBNA2 vector. β-actin was used as loading control. Densitometry analysis is shown in supplemental Figure 1B. (Bii) ICOSL MFI in BJAB and BJABK3 cells was assessed by flow cytometry. The analysis was performed with the FlowJo 10.8.2 portal. One representative experiment of 3 experiments is shown. (Biii) Average ICOSL MFI of 3 experiments by flow cytometry. (Ci-ii) ICOSL mRNA as measured by RT-qPCR in EBNA2-transfected B-lymphoma cell lines. The experiments were repeated at least 3 times and in triplicates each time. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene to normalize the fold change of ICOSL. The fold change was calculated with the formula 2^−ΔΔct. The statistical analysis was performed using a 2-tailed unpaired t test; ∗∗P < .01, ∗∗∗P < .001.