Supplemental Figure Legends
Supplementary Figure 1. Identification and purification of the fusion proteins. (A) Western blot analysis using an anti-His monoclonal antibody showed that the fusion proteins were reactive with the antibody, confirming the presence of the fusion proteins. (B) The size and purity of the recombinant proteins used in this study were confirmed using SDS-PAGE under reducing conditions and Coomassie blue staining.
Supplementary Figure 2. Expression of BAFF-R, TACI and BCMA mRNA in various types of tumor cell lines. The levels of BAFF-R, TACI and BCMA mRNA were analyzed by RT-PCR in Raji, Namalwa, Daudi, JeKo-1, THP-1 and Jurkat cells. Human β-actin was used as a control.
Supplementary Figure 3. mBAFF mediates the specific binding and internalization of PinX1/N-containing fusion proteins into BAFF receptor-positive cells but not BAFF receptor-negative cells. The BAFF receptor-positive cells (Raji, Namalwa and Daudi) and BAFF receptor-negative cells (Jurkat) were treated with 250 nM fusion proteins for 1 h. The cells were then adhered onto a microscope slide and fixed in 4% paraformaldehyde followed by a brief rinse with PBS. Subsequently, the cells were incubated with an anti-His monoclonal antibody. After a brief wash with PBS, cells were incubated with FITC-conjugated goat anti-mouse IgG. After washing with PBS, slides were mounted in mounting medium and then analyzed by laser scanning confocal microscopy.
Supplementary Figure 4. Cell growth inhibition assay with the PinX1/N-containing fusion proteins. (A) Cell proliferation assay was performed using [3H]Thymidine incorporation method. The BAFF receptor-expressing cells (Raji, Namalwa and Daudi) were seeded into a 96-well flat-bottom plate (1×104 cells per well), and treated in triplicate with the fusion proteins at various concentrations. Then the cells were incubated for 5 days, and [3H]-thymidine was added to the wells (1 μCi/well) during the last 16 h of incubation. Subsequently, the cells were collected on glass fiber filters, washed, dried, and counted using standard scintillation methods, and the inhibition of proliferation percentages were calculated. (B) Cell proliferation assay was performed in BAFF receptor-negative Jurkat cells as described above. (C) Blocking assay was conducted to confirm the importance of the fused mBAFF for the activity of PinX1-containing fusion proteins. Raji cells were pretreated with 10 μg/mL of mBAFF for 1 h, followed by treatment with 250 nM of the fusion proteins in quadruplicate wells. The cells were then incubated for 5 days, and proliferation assays measuring [3H]-thymidine incorporation were conducted. DHFR, which does not bind BAFF receptors, serves as a negative control. All data are expressed as mean±SE.
Supplementary Figure 5. Apoptosis assay with the PinX1/N-containing fusion proteins. Raji and Jurkat cells were treated in triplicate with 250 nM fusion proteins for 5 days, and apoptosis were then measured using flow cytometry after staining cells with annexin V-PI. Specific apoptosis in comparison with control cell death was calculated and presented as percent of control. The results are expressed as mean±SE. (A) and (B) Apoptosis assay with the PinX1-containing fusion proteins. (C) and (D) Apoptosis assay with the PinX1N-containing fusion proteins.