Figure 3
Figure 3. Pax5−/− huPax5 pro-/pre-B-cell clone 4 can be induced to develop into biphenotypic cells by graded huPax5 expression induced by different levels of doxycycline in vitro. (A) Experimental overview: Pax5−/− huPax5 pro-/pre-B-cell clone 4 was induced to express huPax5 by different levels of doxycycline (0-1000 ng/mL) in IL-7/OP9 for 3 days in vitro and was subsequently cultivated in SCF/Flt3l/OP9 with the same levels of doxycycline for 23 days. (B) Quantitative RT-PCR for huPax5 (i) and CD19 (ii) mRNA levels of Pax5−/− huPax5 pro-/pre-B-cell clone 4 induced by different levels of doxycycline (0-1000 ng/mL) in IL-7/OP9 in vitro. Results were normalized against GAPDH expression and plotted relative to control Pax5−/− rtTA pro-/pre-B cells. Each bar represents the mean ± SEM (error bars) of 3 individual experiments. (C) Western blot analysis with a Pax5-specific antibody of whole cellular lysates of Pax5−/− huPax5 pro-/pre-B-cell clone 4 before and 3 days after graded doxycycline administration (0-1000ng/mL) in vitro in comparison to wild-type pre-B-I cells and Pax5−/− rtTA pro-/pre-B cells. β-actin–specific antibody was used as a loading control. (D) Representative FACS analysis (n = 3) of CD19 surface expression of Pax5−/−huPax5 pro-/pre-B-cell clone 4 induced by different levels of doxycycline (0-1000 ng/mL) in vitro. CD19 surface expression was detected at doxycycline levels more than 100 ng/mL. (E) Growth curves of Pax5−/−huPax5 pro-/pre-B-cell clone 4 induced to express huPax5 by different levels of doxycycline (0, 10, 300, and 1000 ng/mL) in IL-7/OP9 that were subsequently cultivated in SCF/Flt3l/OP9 (d0) at the same doxycycline concentrations for 23 days. (F) Representative FACS analysis (n = 3) of B220, CD19, and CD11b surface expression of Pax5−/−huPax5 pro-/pre-B-cell clone 4 cultivated for 4 days in SCF/Flt3l/OP9 at different levels of doxycycline (0, 10, 300, and 1000 ng/mL) in vitro. The numbers represent percentages of cells. B220+/CD19−/CD11b+ biphenotypic cells develop at low doxycycline concentrations (0-300 ng/mL).

Pax5−/− huPax5 pro-/pre-B-cell clone 4 can be induced to develop into biphenotypic cells by graded huPax5 expression induced by different levels of doxycycline in vitro. (A) Experimental overview: Pax5−/− huPax5 pro-/pre-B-cell clone 4 was induced to express huPax5 by different levels of doxycycline (0-1000 ng/mL) in IL-7/OP9 for 3 days in vitro and was subsequently cultivated in SCF/Flt3l/OP9 with the same levels of doxycycline for 23 days. (B) Quantitative RT-PCR for huPax5 (i) and CD19 (ii) mRNA levels of Pax5−/− huPax5 pro-/pre-B-cell clone 4 induced by different levels of doxycycline (0-1000 ng/mL) in IL-7/OP9 in vitro. Results were normalized against GAPDH expression and plotted relative to control Pax5−/− rtTA pro-/pre-B cells. Each bar represents the mean ± SEM (error bars) of 3 individual experiments. (C) Western blot analysis with a Pax5-specific antibody of whole cellular lysates of Pax5−/− huPax5 pro-/pre-B-cell clone 4 before and 3 days after graded doxycycline administration (0-1000ng/mL) in vitro in comparison to wild-type pre-B-I cells and Pax5−/− rtTA pro-/pre-B cells. β-actin–specific antibody was used as a loading control. (D) Representative FACS analysis (n = 3) of CD19 surface expression of Pax5−/−huPax5 pro-/pre-B-cell clone 4 induced by different levels of doxycycline (0-1000 ng/mL) in vitro. CD19 surface expression was detected at doxycycline levels more than 100 ng/mL. (E) Growth curves of Pax5−/−huPax5 pro-/pre-B-cell clone 4 induced to express huPax5 by different levels of doxycycline (0, 10, 300, and 1000 ng/mL) in IL-7/OP9 that were subsequently cultivated in SCF/Flt3l/OP9 (d0) at the same doxycycline concentrations for 23 days. (F) Representative FACS analysis (n = 3) of B220, CD19, and CD11b surface expression of Pax5−/−huPax5 pro-/pre-B-cell clone 4 cultivated for 4 days in SCF/Flt3l/OP9 at different levels of doxycycline (0, 10, 300, and 1000 ng/mL) in vitro. The numbers represent percentages of cells. B220+/CD19−/CD11b+ biphenotypic cells develop at low doxycycline concentrations (0-300 ng/mL).

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