Figure 6. Dysregulated gene expression in Ccnd3−/− progenitors includes Ig V genes. (A) Heat map representing differential expression of genes in pro–B cells from WT and Ccnd3−/− bone marrow, grouped based on hierarchical clustering, n = 2. (B) qPCR analysis of the expression of Ccnd2 and Ccnd3 in pro–B cells, shown as the fold change in Ccnd3−/− relative to WT. (C) qPCR analysis of the expression of Ccne1 and Ccna2 in pro–B cells, shown as the fold change in WT relative to Ccnd3−/−. Both B and C represent three independent experiments, shown as mean ± SD. (D–G) qPCR analysis of the expression of Igkv1-117 (D), Igh-VJ558 (E), Iglv1 (F), and Igk germline (G) in WT and Ccnd3−/− pro–B and small pre–B cells, normalized to β2 microglobulin (B2m) expression and shown as mean ± SD. Data are representative of three independent experiments. (H) qPCR analysis of Igk rearrangements involving Igkj1, normalized to amplification of Eκi, in WT and Ccnd3−/− pro–B and small pre–B cells and shown as mean ± SD. Data are representative of three independent experiments. (I) Distribution of VH family usage in WT and Ccnd3−/− large pre–B cells. Results represent sequences derived from three independent experiments (WT = 173 and Ccnd3−/− = 162 sequences). (J) qPCR analysis of the expression of Igkv2-137, Igkv9-120, Igkv1-117, Igkv13-85, Igkv4-70, Igkv12-41, Igkv3-5, and Igkv3-1 (organized as most distal to most proximal) in pro–B cells, presented as the fold change (mean ± SD) in Ccnd3−/− relative to WT, n = 3. (K) qPCR analysis of Igk germline, Igkv1-117, Igh-VJ558, Iglv1, Ccne, and Ccna2 in Ccnd3−/− pro–B cells expressing empty vector or WT cyclin D3 (mean ± SD), presented as in D–G. Data are representative of at least three independent experiments. (L–N) qPCR analysis of Igkv1-117 (L), Ccne (M), and Ccna2 (N) in Ccnd3−/− pro–B cells expressing WT cyclin D3 or chimeric proteins containing the N-terminal or C-terminal region of cyclin D3 (N-D3 and C-D3, respectively). Expression of targets was normalized to corresponding WT, N-D3, or C-D3 expression, standardized to values for WT to facilitate comparisons and presented here as mean ± SD in arbitrary units (A.U.). Data are representative of three independent experiments. (O) Rag2−/− pro–B cells were cultured in IL-7 with or without LY294002 for 24 h and assayed by qPCR for the targets indicated (mean ± SD), presented as in D–G. Data are representative of three independent experiments. (P) Rag2−/− pro–B cells were cultured in IL-7 with or without PD0332991 for 24 h and assayed by qPCR for the targets indicated (mean ± SD) as in O. Data are representative of three independent experiments. †, P < 0.05; §, P < 0.01; *, P < 0.001; ‡, P < 0.0001.
Figure 6.

Dysregulated gene expression in Ccnd3−/− progenitors includes Ig V genes. (A) Heat map representing differential expression of genes in pro–B cells from WT and Ccnd3−/− bone marrow, grouped based on hierarchical clustering, n = 2. (B) qPCR analysis of the expression of Ccnd2 and Ccnd3 in pro–B cells, shown as the fold change in Ccnd3−/− relative to WT. (C) qPCR analysis of the expression of Ccne1 and Ccna2 in pro–B cells, shown as the fold change in WT relative to Ccnd3−/−. Both B and C represent three independent experiments, shown as mean ± SD. (D–G) qPCR analysis of the expression of Igkv1-117 (D), Igh-VJ558 (E), Iglv1 (F), and Igk germline (G) in WT and Ccnd3−/− pro–B and small pre–B cells, normalized to β2 microglobulin (B2m) expression and shown as mean ± SD. Data are representative of three independent experiments. (H) qPCR analysis of Igk rearrangements involving Igkj1, normalized to amplification of Eκi, in WT and Ccnd3−/− pro–B and small pre–B cells and shown as mean ± SD. Data are representative of three independent experiments. (I) Distribution of VH family usage in WT and Ccnd3−/− large pre–B cells. Results represent sequences derived from three independent experiments (WT = 173 and Ccnd3−/− = 162 sequences). (J) qPCR analysis of the expression of Igkv2-137, Igkv9-120, Igkv1-117, Igkv13-85, Igkv4-70, Igkv12-41, Igkv3-5, and Igkv3-1 (organized as most distal to most proximal) in pro–B cells, presented as the fold change (mean ± SD) in Ccnd3−/− relative to WT, n = 3. (K) qPCR analysis of Igk germline, Igkv1-117, Igh-VJ558, Iglv1, Ccne, and Ccna2 in Ccnd3−/− pro–B cells expressing empty vector or WT cyclin D3 (mean ± SD), presented as in D–G. Data are representative of at least three independent experiments. (L–N) qPCR analysis of Igkv1-117 (L), Ccne (M), and Ccna2 (N) in Ccnd3−/− pro–B cells expressing WT cyclin D3 or chimeric proteins containing the N-terminal or C-terminal region of cyclin D3 (N-D3 and C-D3, respectively). Expression of targets was normalized to corresponding WT, N-D3, or C-D3 expression, standardized to values for WT to facilitate comparisons and presented here as mean ± SD in arbitrary units (A.U.). Data are representative of three independent experiments. (O) Rag2−/− pro–B cells were cultured in IL-7 with or without LY294002 for 24 h and assayed by qPCR for the targets indicated (mean ± SD), presented as in D–G. Data are representative of three independent experiments. (P) Rag2−/− pro–B cells were cultured in IL-7 with or without PD0332991 for 24 h and assayed by qPCR for the targets indicated (mean ± SD) as in O. Data are representative of three independent experiments. †, P < 0.05; §, P < 0.01; *, P < 0.001; ‡, P < 0.0001.

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