Figure 3
Figure 3. Differential compartmentalization, immunostimulatory effects, target down-modulation, and cytotoxicity by G3139 and RIT-INP–G3139 in CLL B cells. (A) RIT-INPs mediate the early endosomal compartmentalization of G3139. Purified CLL B cells were incubated with free FAM-G3139 (2μM) or RIT-INP–FAM-G3139 (2μM) for 1 hour. Cells were washed, fixed, and stained with anti–EEA-1 or anti–LAMP-1 antibodies for confocal observation. The white arrows indicate the colocalization (yellow dots). Scale bar indicates 10 μm. (B) Fold changes of costimulatory molecules relative to medium control in CLL B cells. Primary CLL B cells were incubated in the presence of free G3139 (1μM), and RIT-INP–G3139 (1μM). The data are based on MFI. Results are shown as means of n = 10 independent experiments. (C) Effect of RIT-INP–G3139 on Bcl-2 protein level. Top panel is a comparison of relative Bcl-2 protein level (n = 5, mean ± SEM). Primary CLL B cells were incubated with free G3139 or HER-INP- or RIT-INP–formulated G3139 and G3622 at 2μM for 48 hours. Average Western blot band intensities were determined by densitometry and data are presented as relative percentages compared with untreated cell controls. Bottom panel is a presentative Western blot analysis of Bcl-2 protein in CLL B cells. (D) Relative percentage of CLL B-cell viability normalized to medium controls. CLL B cells were treated with various conditions at 37°C for 48 hours. The percentage of viable cells was determined by annexin V/propidium iodide staining and was analyzed by flow cytometry (n = 5, mean ± SEM). (E) Improved cytotoxicity of fludarabine after treatment by G3139-loaded RIT-INPs. Relative cell viability was determined by propidium iodide staining of free G3139 and various G3139-containing INPs at 1μM for 24 hours, followed by fludarabine (1μM) for another 48 hours. Results are presented as means of n = 4 independent experiments. (F) Induction of NF-κB–binding activity detected by the EMSA assay. CLL B cells were incubated with free G3139 and INP-loaded G3139 at 1μM for 4 hours. The CLL cells were stimulated with 500 ng/mL of CD40L for 1 hour as a positive control. (G) Western blot analysis of NF-κB (p65) phosphoration. CLL B cells were incubated with free G3139 and INP-loaded G3139 at 1μM for 3 hours, followed by lysis for Western blot analysis. (H) Differential cytokine inductions of IL-6, TNF-α, and IFN-γ on CLL B cells. Primary CLL B cells were treated under the indicated conditions for 48 hours. Supernatants were collected for ELISA analysis.

Differential compartmentalization, immunostimulatory effects, target down-modulation, and cytotoxicity by G3139 and RIT-INP–G3139 in CLL B cells. (A) RIT-INPs mediate the early endosomal compartmentalization of G3139. Purified CLL B cells were incubated with free FAM-G3139 (2μM) or RIT-INP–FAM-G3139 (2μM) for 1 hour. Cells were washed, fixed, and stained with anti–EEA-1 or anti–LAMP-1 antibodies for confocal observation. The white arrows indicate the colocalization (yellow dots). Scale bar indicates 10 μm. (B) Fold changes of costimulatory molecules relative to medium control in CLL B cells. Primary CLL B cells were incubated in the presence of free G3139 (1μM), and RIT-INP–G3139 (1μM). The data are based on MFI. Results are shown as means of n = 10 independent experiments. (C) Effect of RIT-INP–G3139 on Bcl-2 protein level. Top panel is a comparison of relative Bcl-2 protein level (n = 5, mean ± SEM). Primary CLL B cells were incubated with free G3139 or HER-INP- or RIT-INP–formulated G3139 and G3622 at 2μM for 48 hours. Average Western blot band intensities were determined by densitometry and data are presented as relative percentages compared with untreated cell controls. Bottom panel is a presentative Western blot analysis of Bcl-2 protein in CLL B cells. (D) Relative percentage of CLL B-cell viability normalized to medium controls. CLL B cells were treated with various conditions at 37°C for 48 hours. The percentage of viable cells was determined by annexin V/propidium iodide staining and was analyzed by flow cytometry (n = 5, mean ± SEM). (E) Improved cytotoxicity of fludarabine after treatment by G3139-loaded RIT-INPs. Relative cell viability was determined by propidium iodide staining of free G3139 and various G3139-containing INPs at 1μM for 24 hours, followed by fludarabine (1μM) for another 48 hours. Results are presented as means of n = 4 independent experiments. (F) Induction of NF-κB–binding activity detected by the EMSA assay. CLL B cells were incubated with free G3139 and INP-loaded G3139 at 1μM for 4 hours. The CLL cells were stimulated with 500 ng/mL of CD40L for 1 hour as a positive control. (G) Western blot analysis of NF-κB (p65) phosphoration. CLL B cells were incubated with free G3139 and INP-loaded G3139 at 1μM for 3 hours, followed by lysis for Western blot analysis. (H) Differential cytokine inductions of IL-6, TNF-α, and IFN-γ on CLL B cells. Primary CLL B cells were treated under the indicated conditions for 48 hours. Supernatants were collected for ELISA analysis.

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