Evaluation of in vivo therapeutic efficacy of RIT-INP–G3139. (A) Enhanced Bcl-2 down-regulation of RIT-INP–G3139 in Raji cells. Raji cells were treated with free G3139 (2μM) or various formulated G3139 (2μM) and G3622 (2μM) for 48 hours. Cells were collected and lysed for Western blot analysis. (B) Relative percentage of Raji cell viability of RIT-INP–G3139 normalized to medium control. The percentage of viable cells was determined by annexin V/propidium iodide staining and was analyzed by flow cytometry. Results are presented as means of n = 3 independent experiments. (C) Survival curve for NOD-SCID Raji mice treated with various formulations of G3139 at 5 mg/kg. NOD-SCID mice engrafted with 2 × 10⁶ Raji cells on day 0. The 10-day treatment was initiated via IP injection on day 4. For another in vivo study (D-G), mice were engrafted by Raji cells and treated with free G3139 (5 mg/kg), RIT-INP–G3139 (5 mg/kg), or HER-INP–G3139 (5 mg/kg) on day 10 after inoculation and total 3 treatments were given for each group. On day 15, mice were killed for analysis. (D) Percentage of CD20⁺ cells from BM assessed by flow cytometry. (E) Immunohistochemical staining of Bcl-2 on BM from the Raji xenograft model. The arrows indicate the intracellular Bcl-2 stained Raji cells (brown color) among the control and treated groups. (F) Fold changes of costimulatory molecules (CD40 and CD86) in cells from BM. Data were normalized to the PBS-treated group (n = 4, mean ± SEM). (G) Cytokine production of IL-6 (n = 4) and IFN-γ (n = 4) in serum from mice treated with PBS, free G3139 (5 mg/kg), and RIT-INP–G3139 (5 mg/kg) or HER-INP–G3139 (5 mg/kg). Cytokine release was determined by ELISA.