Figure 5.
RelB protects ABC and GCB DLBCL cells from induction of apoptosis upon genotoxic treatment. (A) RelB protein levels (top) and RelB DNA-binding activity (bottom) are efficiently knocked down by stable RNA interference in ABC and GCB DLBCL cell lines. Whole cell extracts from MD901 (ABC), K231 (GCB), and OCI-Ly8 (GCB) cell lines transduced with lentiviruses encoding either a short hairpin RNA (shRNA) targeting RelB (shRNA RelB) or a scrambled control (shRNA control) were analyzed by using immunoblotting (top) and by EMSA (bottom) for the indicated proteins. (B) RelB knockdown induces ABC and GCB DLBCL cell apoptosis upon doxorubicin (top), etoposide (middle), and camptothecin (bottom) treatment. MD901, K231, and OCI-Ly8 cell lines transduced as in panel A were treated with the indicated genotoxic agent for 16 hours and monitored for apoptosis by annexin V/Allophycocyanin (APC) and DAPI staining followed by FACS analysis. Error bars are means ± standard deviation of 3 independent experiments. P values by unpaired Student t test , *P < .05, **P < .01, ***P < .001. Whole cell extracts of MD901 cells transduced as in panel A were treated with doxorubicin 2 µM for the indicated periods of time and analyzed by using immunoblotting for cleaved caspase 3 (C) and γ-H2AX (D). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (panel C) and β-actin (panel D) were also analyzed, n = 3. (E) RelB knockdown induces γ-H2AX foci in DLBCL cells upon doxorubicin treatment. Left: percentage of γH2AX foci-positive MD901 cells. Right: γH2AX foci count mean in response to doxorubicin normalized to foci count mean of untreated MD901 cells. Data of a representative experiment are shown. (F) RelB knockdown decreases cIAP2 expression at mRNA level in ABC and GCB DLBCL cell lines. Quantitative reverse transcription polymerase chain reaction was performed with specific primer pairs for cIAP2 using total RNAs prepared from three DLBCL cell lines (1 ABC and 2 GCB) upon RelB knockdown as in panel A. Results are means ± standard error of the mean of 3 independent experiments for each cell line normalized to the level of HPRT mRNA. P values by unpaired Student t test, ***P < .001, ****P < .0001. (G) RelB knockdown decreases cIAP2 protein expression levels in ABC and GCB DLBCL cell lines. Whole cell extracts prepared as in panel A were analyzed according to the immunoblotting for the indicated proteins, n = 2. (H) RelB activation as evaluated by EMSA is associated with increased cIAP2 protein expression in patients with DLBCL. Whole cell extracts from 24 R-CHOP–treated patients with DLBCL of the EMSA study were analyzed for cIAP2 protein expression levels by immunoblotting. Left: 1 representative immunoblot is presented. Right: cIAP2 protein expression levels normalized to β-actin, n = 24. P values by Pearson correlation test, **P < .01.

RelB protects ABC and GCB DLBCL cells from induction of apoptosis upon genotoxic treatment. (A) RelB protein levels (top) and RelB DNA-binding activity (bottom) are efficiently knocked down by stable RNA interference in ABC and GCB DLBCL cell lines. Whole cell extracts from MD901 (ABC), K231 (GCB), and OCI-Ly8 (GCB) cell lines transduced with lentiviruses encoding either a short hairpin RNA (shRNA) targeting RelB (shRNA RelB) or a scrambled control (shRNA control) were analyzed by using immunoblotting (top) and by EMSA (bottom) for the indicated proteins. (B) RelB knockdown induces ABC and GCB DLBCL cell apoptosis upon doxorubicin (top), etoposide (middle), and camptothecin (bottom) treatment. MD901, K231, and OCI-Ly8 cell lines transduced as in panel A were treated with the indicated genotoxic agent for 16 hours and monitored for apoptosis by annexin V/Allophycocyanin (APC) and DAPI staining followed by FACS analysis. Error bars are means ± standard deviation of 3 independent experiments. P values by unpaired Student t test , *P < .05, **P < .01, ***P < .001. Whole cell extracts of MD901 cells transduced as in panel A were treated with doxorubicin 2 µM for the indicated periods of time and analyzed by using immunoblotting for cleaved caspase 3 (C) and γ-H2AX (D). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (panel C) and β-actin (panel D) were also analyzed, n = 3. (E) RelB knockdown induces γ-H2AX foci in DLBCL cells upon doxorubicin treatment. Left: percentage of γH2AX foci-positive MD901 cells. Right: γH2AX foci count mean in response to doxorubicin normalized to foci count mean of untreated MD901 cells. Data of a representative experiment are shown. (F) RelB knockdown decreases cIAP2 expression at mRNA level in ABC and GCB DLBCL cell lines. Quantitative reverse transcription polymerase chain reaction was performed with specific primer pairs for cIAP2 using total RNAs prepared from three DLBCL cell lines (1 ABC and 2 GCB) upon RelB knockdown as in panel A. Results are means ± standard error of the mean of 3 independent experiments for each cell line normalized to the level of HPRT mRNA. P values by unpaired Student t test, ***P < .001, ****P < .0001. (G) RelB knockdown decreases cIAP2 protein expression levels in ABC and GCB DLBCL cell lines. Whole cell extracts prepared as in panel A were analyzed according to the immunoblotting for the indicated proteins, n = 2. (H) RelB activation as evaluated by EMSA is associated with increased cIAP2 protein expression in patients with DLBCL. Whole cell extracts from 24 R-CHOP–treated patients with DLBCL of the EMSA study were analyzed for cIAP2 protein expression levels by immunoblotting. Left: 1 representative immunoblot is presented. Right: cIAP2 protein expression levels normalized to β-actin, n = 24. P values by Pearson correlation test, **P < .01.

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