FOXO3A is highly expressed in cHL. (A) Gene expression profiling (GEP) data were mined and analyzed with help of the GENEVESTIGATOR software. The following data sets were used for analysis: CB and CC (GSE15271, GSE38697, GSE56314); M (GSE1266, GSE45113, GSE17186, GSE64028); PC (GSE12366, GSE45537, GSE56464, GSE5900), cHL (GSE14879). FOXO1: probe set 202724_s_at; FOXO3A: probe set 204131_s_at; FOXO4: probe set 205451_at; FOXO6: probe set 239657_x_at. CB (n = 15); centrocytes (CC; n = 15); memory B cells (M; n = 38); PC (n = 13); microdissected HRS cells of cHL (cHL; n = 4). Data are represented as mean fluorescence ± standard deviation (SD). Means of FOXO3A levels in GC B cells and cHL were compared using an online software (https://www.medcalc.org/calc/comparison_of_means.php; 10.23.2017). ****P < .0001. (B-H) FOXO1 and FOXO3A mRNA and protein expression in CD19⁺ B cells and B- or T-cell–derived cHL cell lines. FOXO1 (B) and FOXO3A (C) mRNA levels were measured by RT-qPCR and expressed as ratio to RPL13A. Data are shown as mean ± SD of 3 independent experiments. FOXO1 and FOXO3A expressions of all cHL cell lines except for FOXO3A in L540 significantly differed from CD19⁺ B cells with P < .01. Statistical significances were analyzed by 2-tailed t tests. (D) FOXO3A/FOXO1 mRNA ratio in B cells and cHL. Data were extracted from experiments shown in panels B and C. (E) FOXO1 and FOXO3A protein expression was measured by immunoblot. The most representative of 3 independent analyses is shown. TUBB served as loading control. (F-G) The immunoblots were quantified using ImageJ 64 software. FOXO1 (F) and FOXO3A (G) values were normalized to TUBB. Data are shown as mean ± SD of all 3 experiments and values for KM-H2 were set to 1. (H) The apparent FOXO3A-to-FOXO1 protein ratio for each sample was calculated as: (FOXO3A/TUBB)/(FOXO1/TUBB). The apparent ratio does not represent the molar FOXO3A-to-FOXO1 ratio but permits to compare relative expression of FOXO proteins in different samples.