Figure 7.
Notch1 hyperactivation promotes proliferation of CLL cells. (A) Representative flow cytometric profiles are shown of a BrdU incorporation assay performed on splenocytes isolated from IgH.TEμ;control and IgH.TEμ;RNICΔ/+ mb1-Cre mice. BrdU was injected intraperitoneally 48 hours before analysis. Upper contour plots show CD19 vs B220 expression gated on live cells. Lower panels depict BrdU vs DAPI expression gated on CD19+B220− cells from the upper panels. S, S-phase; G0-G1, G0- or G1-phase; G2-M, G2- or M-phase. (B) Quantification of relative percentages of CLL cells (CD19+B220−) and B cells (CD19+B220+) in S-phase isolated from IgH.TEμ;control (IgH.TEμ;RNIClox/+ n = 4 and IgH.TEμmb1-Cre n = 3; pooled n = 7, blue circles) and IgH.TEμ;RNICΔ/+ mb1-Cre mice (n = 7, red filled squares) are plotted. The graph represents data pooled from 2 independent experiments. P values were calculated using unpaired Student t test: ****P < .0001. (C) Representative flow cytometric profiles of a BrdU incorporation assay performed on splenocytes isolated from IgH.TEμ;control (IgH.TEμ;RNIClox/+ n = 4 and IgH.TEμCD19-Cre n = 2, pooled) and IgH.TEμ;RNICΔ/+ CD19-Cre mice (n = 8) are shown. BrdU was injected intraperitoneally 48 hours prior to analysis. Upper panels show CD19 vs B220 expression gated on live cells. Lower panels depict BrdU vs DAPI expression gated on CD19+B220− cells from the upper panels. (D) Relative percentages of CLL cells (CD19+B220−) and B cells (CD19+B220+) in S-phase from the IgH.TEμ;controls (n = 6, blue filled circles) and IgH.TEμ;RNICΔ/+ mb1-Cre mice (n = 8, red filled squares) are plotted. The graph represents data pooled from 2 independent experiments. P values were calculated using unpaired Student t test: **P = .0036. (E) Representative immunohistochemical staining for p-histone H3 (p-HH3) of spleen (top) and LN (bottom) sections from 7-month-old WT control, IgH.TEμ, and IgH.TEμ;RNICΔ/+ mb1-Cre mice is shown. Quantification of p-HH3+ stained cells in WT (n = 4), IgH.TEμ (n = 4), and IgH.TEμ;RNICΔ/+ mb1-Cre (n = 3) is depicted on the right.

Notch1 hyperactivation promotes proliferation of CLL cells. (A) Representative flow cytometric profiles are shown of a BrdU incorporation assay performed on splenocytes isolated from IgH.TEμ;control and IgH.TEμ;RNICΔ/+ mb1-Cre mice. BrdU was injected intraperitoneally 48 hours before analysis. Upper contour plots show CD19 vs B220 expression gated on live cells. Lower panels depict BrdU vs DAPI expression gated on CD19+B220− cells from the upper panels. S, S-phase; G0-G1, G0- or G1-phase; G2-M, G2- or M-phase. (B) Quantification of relative percentages of CLL cells (CD19+B220−) and B cells (CD19+B220+) in S-phase isolated from IgH.TEμ;control (IgH.TEμ;RNIClox/+ n = 4 and IgH.TEμmb1-Cre n = 3; pooled n = 7, blue circles) and IgH.TEμ;RNICΔ/+ mb1-Cre mice (n = 7, red filled squares) are plotted. The graph represents data pooled from 2 independent experiments. P values were calculated using unpaired Student t test: ****P < .0001. (C) Representative flow cytometric profiles of a BrdU incorporation assay performed on splenocytes isolated from IgH.TEμ;control (IgH.TEμ;RNIClox/+ n = 4 and IgH.TEμCD19-Cre n = 2, pooled) and IgH.TEμ;RNICΔ/+ CD19-Cre mice (n = 8) are shown. BrdU was injected intraperitoneally 48 hours prior to analysis. Upper panels show CD19 vs B220 expression gated on live cells. Lower panels depict BrdU vs DAPI expression gated on CD19+B220− cells from the upper panels. (D) Relative percentages of CLL cells (CD19+B220−) and B cells (CD19+B220+) in S-phase from the IgH.TEμ;controls (n = 6, blue filled circles) and IgH.TEμ;RNICΔ/+ mb1-Cre mice (n = 8, red filled squares) are plotted. The graph represents data pooled from 2 independent experiments. P values were calculated using unpaired Student t test: **P = .0036. (E) Representative immunohistochemical staining for p-histone H3 (p-HH3) of spleen (top) and LN (bottom) sections from 7-month-old WT control, IgH.TEμ, and IgH.TEμ;RNICΔ/+ mb1-Cre mice is shown. Quantification of p-HH3+ stained cells in WT (n = 4), IgH.TEμ (n = 4), and IgH.TEμ;RNICΔ/+ mb1-Cre (n = 3) is depicted on the right.

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