American Society of Hematology

Figure 5

$Figure 5. CRBN silencing interferes with p21, IKZF1 and IKZF3 expression, and the antiproliferative activity of lenalidomide in CLL cells. (A-B) CLL cells were transfected with CRBN siRNA or nonspecific control siRNA (CTRL) using Amaxa, and cocultured on FibroblastsCD154 with IL-4 and IL-10 for 48 hours, at which point the cells were collected and lyzed for the analysis of CRBN protein expression and β-actin by immunoblot. Data from 2 representative patients are shown in (A) and densitometry analysis quantifying the expression levels of CRBN protein in 3 transfected CLL samples is presented in (B). The expression of CRBN has been normalized to β-actin. *P < .05 (Student t test, mean ± SEM; n = 3). (C-D) CLL cells were transfected with CTRL siRNA or CRBN siRNA as above, and plated on FibroblastsCD154 for 48 hours, at which point increasing doses of lenalidomide were added to the cells. After 5 days of lenalidomide exposure, CLL cells were collected and lysed to monitor for p21 and β-actin protein expression by immunoblot. Data from a representative patient is shown in (C) and densitometry analysis quantifying the expression levels of p21 protein in 3 transfected CLL samples is presented in (D). The expression of p21 has been normalized to β-actin. *P < .05 (Student t test, mean ± SEM; n = 3). (E-F) CLL cells were transfected with CTRL siRNA or CRBN siRNA, cocultured on FibroblastsCD154, and exposed to lenalidomide as above. After 5 days of lenalidomide exposure, the fraction of CLL cells in S-phase of the cell cycle was measured by EdU incorporation and flow cytometry. In (E), data from each patient sample tested are presented, while (F) shows the combined data for all 3 patients presented in (E), after being normalized to CTRL cells. *P < .05, **P < .01 (Student t test, mean ± SEM; n = 3). (G-H) CLL cells were transfected with CTRL siRNA or CRBN siRNA, cocultured on FibroblastsCD154, and exposed to lenalidomide as above. After 24 hours of lenalidomide exposure, CLL cells were collected and lysed to monitor for IKZF1, IKZF3, and β-actin protein expression by immunoblot. Data from a representative patient is shown in (G). Densitometry analysis quantifying the expression levels of IKZF1 and IKZF3 obtained using CLL cells from 3 different patients is presented in (H). The expression of each target protein has been normalized to β-actin, and is expressed relatively to control. *P < .05 (Student t test, mean ± SEM; n = 3).$

CRBN silencing interferes with p21, IKZF1 and IKZF3 expression, and the antiproliferative activity of lenalidomide in CLL cells. (A-B) CLL cells were transfected with CRBN siRNA or nonspecific control siRNA (CTRL) using Amaxa, and cocultured on Fibroblasts_CD154 with IL-4 and IL-10 for 48 hours, at which point the cells were collected and lyzed for the analysis of CRBN protein expression and β-actin by immunoblot. Data from 2 representative patients are shown in (A) and densitometry analysis quantifying the expression levels of CRBN protein in 3 transfected CLL samples is presented in (B). The expression of CRBN has been normalized to β-actin. *P < .05 (Student t test, mean ± SEM; n = 3). (C-D) CLL cells were transfected with CTRL siRNA or CRBN siRNA as above, and plated on Fibroblasts_CD154 for 48 hours, at which point increasing doses of lenalidomide were added to the cells. After 5 days of lenalidomide exposure, CLL cells were collected and lysed to monitor for p21 and β-actin protein expression by immunoblot. Data from a representative patient is shown in (C) and densitometry analysis quantifying the expression levels of p21 protein in 3 transfected CLL samples is presented in (D). The expression of p21 has been normalized to β-actin. *P < .05 (Student t test, mean ± SEM; n = 3). (E-F) CLL cells were transfected with CTRL siRNA or CRBN siRNA, cocultured on Fibroblasts_CD154, and exposed to lenalidomide as above. After 5 days of lenalidomide exposure, the fraction of CLL cells in S-phase of the cell cycle was measured by EdU incorporation and flow cytometry. In (E), data from each patient sample tested are presented, while (F) shows the combined data for all 3 patients presented in (E), after being normalized to CTRL cells. *P < .05, **P < .01 (Student t test, mean ± SEM; n = 3). (G-H) CLL cells were transfected with CTRL siRNA or CRBN siRNA, cocultured on Fibroblasts_CD154, and exposed to lenalidomide as above. After 24 hours of lenalidomide exposure, CLL cells were collected and lysed to monitor for IKZF1, IKZF3, and β-actin protein expression by immunoblot. Data from a representative patient is shown in (G). Densitometry analysis quantifying the expression levels of IKZF1 and IKZF3 obtained using CLL cells from 3 different patients is presented in (H). The expression of each target protein has been normalized to β-actin, and is expressed relatively to control. *P < .05 (Student t test, mean ± SEM; n = 3).

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