Implanted subcutaneously into the right flanks of female SCID mice. When

Implanted subcutaneously into the right flanks of female SCID mice. When the tumor nodules were palpable, the mice were divided randomly into three groups with six mice each and treated with NS, control IgG, or PAb via the tail vein. Control IgG and PAb (200 mg/dose, dissolved in NS) were administered seven times every 2 d in a volume of 100 mL along with the control injection in a volume of 100 mL NS. The tumor volume was observed and the tumor size was determined once every 3 d by caliper MedChemExpress GHRH (1-29) measurement as described previously [20].2D Western BlotThe separated proteins were transferred on PVDF membranes and incubated for 2 h at room temperature with a blocking buffer consisting of TBST (Tris-buffered saline +0.01 Tween 20) and 5 skim milk. The PVDF membranes were dyed with Commassie Blue staining solution for 15 min [0.1 Coomassie Brilliant Blue R-250 (w/v) and 50 methanol (v/v)] and outstanding points were marked as landmarks. The membranes were then decolorized for 1 h in destaining solution [40 methanol (v/v) with 10 acetic acid (v/v)], washed, and incubated with PAb for 1 h atTerminal Deoxynucleotidyl Transferase-mediated dUTP Nick end Labeling (TUNEL) AssayCell apoptosis in vivo was examined by TUNEL assay according to the manufacturer’s instructions (Promega, USA). Three tumors per group were analyzed 48 h after the last treatment.Screening of MM by Polyclonal ImmunoglobulinFigure 3. Inhibition of myeloma cells growth in vitro determined by MTT. (A)The growth of PAb-treated cells was significantly inhibited compared with the control IgG and NS groups, and the inhibitory rates on different concentrations on ARH-77 cells after 48 h were 16.7 , 23.98 , 28.47 , and 56.84 . (B)The similar results were shown in U266 cell line. (C) The PAb did not effect growth of HepG2 cell line. doi:10.1371/journal.pone.0059117.gScreening of MM by Polyclonal ImmunoglobulinFigure 4. PAb-induced apoptosis in myeloma cell lines. Flow cytometric analysis revealed the proportion of sub-G1 phase cells (apoptotic cells) to be 7.3 (NS), 9.9 (control), and 52.1 (PAb). The experiments were repeated at least three times. doi:10.1371/journal.pone.0059117.gStatistical AnalysisSPSS version 13 was used for statistical analysis. The statistical significance of results in all of the experiments was determined by Student’s t-test and analysis of variance. The findings were regarded as significant if P,0.05.and subjected to in-gel digestion followed by peptide mass fingerprinting for protein identification. Figure 2C shows the identification of Spot No.1 as an example. The results of antigen identification are summarized in the Appendix, Table 24786787 1.Inhibitory Effect of PAb on ARH-77 Cell Proliferation Results Production and Characterization of PAbTo investigate the possibility of vaccination of rabbits with ARH-77, two rabbits were inoculated with ARH-77 cells to produce polyclonal antibody. PAb was Itacitinib tested for its ability to bind MM cell lines (Fig. 1A). The binding of ARH-77 by PAb differed by 3- to 10-fold from control IgG. The binding was dosedependent, with dilutions of 1:2,000 and 1:5,000 showing greater binding to ARH-77 than dilutions of 1:10,000 or 1:20,000. As to the antigens recognized by PAb, we further performed Western blot, flow cytometric assay, and immunofluorescence studies. ARH-77 cell lysates were probed with either PAb or control IgG on Western blots. Multiple bands (Fig. 1B) were recognized by PAb but not by the control IgG. Immunofluorescence.Implanted subcutaneously into the right flanks of female SCID mice. When the tumor nodules were palpable, the mice were divided randomly into three groups with six mice each and treated with NS, control IgG, or PAb via the tail vein. Control IgG and PAb (200 mg/dose, dissolved in NS) were administered seven times every 2 d in a volume of 100 mL along with the control injection in a volume of 100 mL NS. The tumor volume was observed and the tumor size was determined once every 3 d by caliper measurement as described previously [20].2D Western BlotThe separated proteins were transferred on PVDF membranes and incubated for 2 h at room temperature with a blocking buffer consisting of TBST (Tris-buffered saline +0.01 Tween 20) and 5 skim milk. The PVDF membranes were dyed with Commassie Blue staining solution for 15 min [0.1 Coomassie Brilliant Blue R-250 (w/v) and 50 methanol (v/v)] and outstanding points were marked as landmarks. The membranes were then decolorized for 1 h in destaining solution [40 methanol (v/v) with 10 acetic acid (v/v)], washed, and incubated with PAb for 1 h atTerminal Deoxynucleotidyl Transferase-mediated dUTP Nick end Labeling (TUNEL) AssayCell apoptosis in vivo was examined by TUNEL assay according to the manufacturer’s instructions (Promega, USA). Three tumors per group were analyzed 48 h after the last treatment.Screening of MM by Polyclonal ImmunoglobulinFigure 3. Inhibition of myeloma cells growth in vitro determined by MTT. (A)The growth of PAb-treated cells was significantly inhibited compared with the control IgG and NS groups, and the inhibitory rates on different concentrations on ARH-77 cells after 48 h were 16.7 , 23.98 , 28.47 , and 56.84 . (B)The similar results were shown in U266 cell line. (C) The PAb did not effect growth of HepG2 cell line. doi:10.1371/journal.pone.0059117.gScreening of MM by Polyclonal ImmunoglobulinFigure 4. PAb-induced apoptosis in myeloma cell lines. Flow cytometric analysis revealed the proportion of sub-G1 phase cells (apoptotic cells) to be 7.3 (NS), 9.9 (control), and 52.1 (PAb). The experiments were repeated at least three times. doi:10.1371/journal.pone.0059117.gStatistical AnalysisSPSS version 13 was used for statistical analysis. The statistical significance of results in all of the experiments was determined by Student’s t-test and analysis of variance. The findings were regarded as significant if P,0.05.and subjected to in-gel digestion followed by peptide mass fingerprinting for protein identification. Figure 2C shows the identification of Spot No.1 as an example. The results of antigen identification are summarized in the Appendix, Table 24786787 1.Inhibitory Effect of PAb on ARH-77 Cell Proliferation Results Production and Characterization of PAbTo investigate the possibility of vaccination of rabbits with ARH-77, two rabbits were inoculated with ARH-77 cells to produce polyclonal antibody. PAb was tested for its ability to bind MM cell lines (Fig. 1A). The binding of ARH-77 by PAb differed by 3- to 10-fold from control IgG. The binding was dosedependent, with dilutions of 1:2,000 and 1:5,000 showing greater binding to ARH-77 than dilutions of 1:10,000 or 1:20,000. As to the antigens recognized by PAb, we further performed Western blot, flow cytometric assay, and immunofluorescence studies. ARH-77 cell lysates were probed with either PAb or control IgG on Western blots. Multiple bands (Fig. 1B) were recognized by PAb but not by the control IgG. Immunofluorescence.

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