American Society of Hematology

Figure 1

$Figure 1. Identification of FOXP1-regulated genes in primary human B cells and DLBCL cell lines by GEM analysis. To identify FOXP1-regulated genes, we conducted GEM analysis upon retroviral overexpression or RNA interference–mediated silencing of FOXP1 in primary B cells and DLBCL cell lines. (A-B) Expression levels of FOXP1 in primary B cells (A) and DLBCL cell lines (B) upon retroviral transduction or siRNA transfection. (A) Human primary B cells were transduced with FOXP1-IRES-YFP or ctrl-IRES-YFP and cultured with CD40L-L cells. (Left) Three days after transduction, YFP-positive fractions were sorted, RNA was isolated, and gene expression levels were analyzed by qRT-PCR. Expression levels were normalized to expression levels in empty vector–transduced cells. (Right) Three days after transduction, cell lysates were harvested and immunoblotted for FOXP1. β-Tubulin was used as a loading control. (B) DLBCL cell lines were transfected with siRNA against FOXP1. (Left) One day after nucleofection, RNA was isolated and gene expression levels were analyzed by qRT-PCR. Expression levels were normalized to expression levels in control siRNA–transduced cells. (Right) Two days after nucleofection with siRNA against FOXP1, cell lysates were harvested and immunoblotted for FOXP1. β-Tubulin was used as a loading control. Asterisk (*) indicates a nonspecific background band. (C) Expression of the proapoptotic genes that were significantly and reproducibly regulated by FOXP1 in microarray analysis of primary B cells and DLBCL cell lines. Data are presented as z-scores calculated within samples of each cell line. The gray squares indicate expression beneath the threshold value (= no expression). The lower panel shows the mean relative expression values of a gene set composed of the 7 proapoptotic genes. (D) Kaplan-Meier plots of the OS of 498 DLBCL patients treated with rituximab-CHOP therapy,37 stratified in 2 groups by expression of the 7 FOXP1-repressed proapoptotic genes. (Left) OS of DLBCL patients stratified in 2 groups by k-means clustering of the 7 genes. The 5-year OS is 72% for the patient group with overall higher (group 0; red) vs 47% in the group with overall lower (group 1; blue) expression of the 7 genes. The color bar displays the z-scores (blue = low, red = high) of each of the 7 proapoptotic genes (from top to bottom): TP63, HRK, EAF2, TP53INP1, AIM2, RASSF6, and BIK. (Right) OS of DLBCL patients stratified in 2 equal-sized groups (separated at the median) by ranking the patients according to their mean z-score of the gene set. The 5-year OS is 72% in the high-expressing (blue) vs 49% in the low-expressing (red) group. The heatmap displays the z-scores (green = low, red = high) of each of the 7 proapoptotic genes (from top to bottom): TP63, RASSF6, BIK, HRK, EAF2, TP53INP1, and AIM. The blue-red bar displays the mean z-scores (blue is low; red is high) for the gene set, according to which the patients were ranked.$

Identification of FOXP1-regulated genes in primary human B cells and DLBCL cell lines by GEM analysis. To identify FOXP1-regulated genes, we conducted GEM analysis upon retroviral overexpression or RNA interference–mediated silencing of FOXP1 in primary B cells and DLBCL cell lines. (A-B) Expression levels of FOXP1 in primary B cells (A) and DLBCL cell lines (B) upon retroviral transduction or siRNA transfection. (A) Human primary B cells were transduced with FOXP1-IRES-YFP or ctrl-IRES-YFP and cultured with CD40L-L cells. (Left) Three days after transduction, YFP-positive fractions were sorted, RNA was isolated, and gene expression levels were analyzed by qRT-PCR. Expression levels were normalized to expression levels in empty vector–transduced cells. (Right) Three days after transduction, cell lysates were harvested and immunoblotted for FOXP1. β-Tubulin was used as a loading control. (B) DLBCL cell lines were transfected with siRNA against FOXP1. (Left) One day after nucleofection, RNA was isolated and gene expression levels were analyzed by qRT-PCR. Expression levels were normalized to expression levels in control siRNA–transduced cells. (Right) Two days after nucleofection with siRNA against FOXP1, cell lysates were harvested and immunoblotted for FOXP1. β-Tubulin was used as a loading control. Asterisk (*) indicates a nonspecific background band. (C) Expression of the proapoptotic genes that were significantly and reproducibly regulated by FOXP1 in microarray analysis of primary B cells and DLBCL cell lines. Data are presented as z-scores calculated within samples of each cell line. The gray squares indicate expression beneath the threshold value (= no expression). The lower panel shows the mean relative expression values of a gene set composed of the 7 proapoptotic genes. (D) Kaplan-Meier plots of the OS of 498 DLBCL patients treated with rituximab-CHOP therapy,³⁷ stratified in 2 groups by expression of the 7 FOXP1-repressed proapoptotic genes. (Left) OS of DLBCL patients stratified in 2 groups by k-means clustering of the 7 genes. The 5-year OS is 72% for the patient group with overall higher (group 0; red) vs 47% in the group with overall lower (group 1; blue) expression of the 7 genes. The color bar displays the z-scores (blue = low, red = high) of each of the 7 proapoptotic genes (from top to bottom): TP63, HRK, EAF2, TP53INP1, AIM2, RASSF6, and BIK. (Right) OS of DLBCL patients stratified in 2 equal-sized groups (separated at the median) by ranking the patients according to their mean z-score of the gene set. The 5-year OS is 72% in the high-expressing (blue) vs 49% in the low-expressing (red) group. The heatmap displays the z-scores (green = low, red = high) of each of the 7 proapoptotic genes (from top to bottom): TP63, RASSF6, BIK, HRK, EAF2, TP53INP1, and AIM. The blue-red bar displays the mean z-scores (blue is low; red is high) for the gene set, according to which the patients were ranked.

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