Figure 1
Figure 1. TLR9-driven immunostimulation of free G3139 is associated with limited modulation of Bcl-2 expression. (A) Effect of free G3139 on Bcl-2 and Mcl-1 protein levels. Top panel shows 2 representative Western blot results of n = 10 B-CLL patient cells. Primary CLL B cells were incubated with 1, 2, and 5μM G3139 for 48 hours and then collected and lysed for Western blot analysis. Bottom panel shows average Western blot band intensities determined by densitometry. Data are presented as relative percentages compared with untreated cell controls (for Bcl-2, n = 10, mean ± SEM; for Mcl-1, n = 4, mean ± SEM). (B) Relative CLL B-cell viability normalized to medium control, determined by annexin V/propidium iodide staining (n = 10). Primary CLL B cells were incubated with 1, 2, and 5μM G3139 for 48 hours. Relative cell viability was defined as the percentage of annexin V− and propidium iodide− cells relative to the untreated control group. (C) Fold changes of surface markers relative to medium controls in CLL B cells after G3139 treatment. Primary CLL B cells were incubated in the presence of 1, 2, and 5μM G3139. After 48 hours, expressions of CD40, CD80, and CD86 were measured by flow cytometry. The data are presented according to MFI (n = 10, mean ± SEM). (D) Effect of knock-down of TLR9 on the immunostimulatory properties of G3139. Top panel is the fold changes of surface markers relative to medium controls after TLR9 down-regulation by siRNA. Primary CLL B cells were transfected by TLR 9 siRNA using electroporation and incubated for 48 hours, followed by further treatment with 1μM G3139. After 24 hours, the expressions of CD40, CD80, and CD86 were measured by flow cytometry. The data are presented as MFI (n = 4, mean ± SEM). Variable cell death (5%-20%) after transfections was observed. Bottom panel is a representative Western blot showing down-regulation of TLR9 expression by siRNA. (E) Subcellular distribution analysis of TLR9 in CLL B cells. Purified CLL B cells were fixed and stained intracellularly with anti-TLR9, anti–EEA-1, or anti–LAMP-1 antibodies as described. Images were acquired using an Olympus FV1000 confocal microscope. Scale bar indicates 10 μm. The white arrows indicate colocalization (yellow dots).

TLR9-driven immunostimulation of free G3139 is associated with limited modulation of Bcl-2 expression. (A) Effect of free G3139 on Bcl-2 and Mcl-1 protein levels. Top panel shows 2 representative Western blot results of n = 10 B-CLL patient cells. Primary CLL B cells were incubated with 1, 2, and 5μM G3139 for 48 hours and then collected and lysed for Western blot analysis. Bottom panel shows average Western blot band intensities determined by densitometry. Data are presented as relative percentages compared with untreated cell controls (for Bcl-2, n = 10, mean ± SEM; for Mcl-1, n = 4, mean ± SEM). (B) Relative CLL B-cell viability normalized to medium control, determined by annexin V/propidium iodide staining (n = 10). Primary CLL B cells were incubated with 1, 2, and 5μM G3139 for 48 hours. Relative cell viability was defined as the percentage of annexin V− and propidium iodide− cells relative to the untreated control group. (C) Fold changes of surface markers relative to medium controls in CLL B cells after G3139 treatment. Primary CLL B cells were incubated in the presence of 1, 2, and 5μM G3139. After 48 hours, expressions of CD40, CD80, and CD86 were measured by flow cytometry. The data are presented according to MFI (n = 10, mean ± SEM). (D) Effect of knock-down of TLR9 on the immunostimulatory properties of G3139. Top panel is the fold changes of surface markers relative to medium controls after TLR9 down-regulation by siRNA. Primary CLL B cells were transfected by TLR 9 siRNA using electroporation and incubated for 48 hours, followed by further treatment with 1μM G3139. After 24 hours, the expressions of CD40, CD80, and CD86 were measured by flow cytometry. The data are presented as MFI (n = 4, mean ± SEM). Variable cell death (5%-20%) after transfections was observed. Bottom panel is a representative Western blot showing down-regulation of TLR9 expression by siRNA. (E) Subcellular distribution analysis of TLR9 in CLL B cells. Purified CLL B cells were fixed and stained intracellularly with anti-TLR9, anti–EEA-1, or anti–LAMP-1 antibodies as described. Images were acquired using an Olympus FV1000 confocal microscope. Scale bar indicates 10 μm. The white arrows indicate colocalization (yellow dots).

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