Supplementary Figure 6: BCR signaling in VH12Vκ4-transgenic B-1a cells.
(a) Intracellular Ca2+ flux in VH12/Vκ4 transgenic wild-type and DKO splenic B-1a cells, as measured by the change in fluorescence emission of a Ca2+ sensor dye after addition (arrow) of a goat anti-mouse IgM F(ab’)2 fragment. (b) Scatter plot of gene expression differences between ex vivo sorted VH12/Vκ4 transgenic wild-type and DKO peritoneal B-1a cells. Genes encoding components of the BCR signaling pathway (GO:0050853 “B cell receptor signaling pathway” plus manually added genes) are highlighted in the left panel, while genes implicated in B-1a cell development and homeostasis as well as genes encoding the α- and β-chains of the IL-5, IL-3 and GM-CSF receptor family are indicated in the right panel. Genes that are discussed in the text are marked in red. RPM, reads per million mapped sequence reads. (c) Expression of the immunoglobulin μ-heavy chain (Ighm) mRNA (RNA-seq; left) and the cell-surface IgM protein (right) is shown for peritoneal B-1a cells from VH12/Vκ4 transgenic wild-type and DKO mice. The Ighm mRNA data were determined by two independent RNA-seq experiments per cell type, and error bars indicate s.e.m. * P < 0.05 as determined by the Student’s t-test. TPM, transcripts per million. (d) Peritoneal cells from VH12/Vκ4 transgenic DKO and wild-type mice as well as peritoneal B-2 cells from wild-type mice were stained on ice for CD19, B220 and CD5, fixed, permeabilized and stained intracellularly (ic) for IgM and phosphorylated epitopes of the indicated signal transducers. Overlay contour plots displaying IgM expression and the phosphorylation status of the indicated BCR signaling components are shown for polyclonal B-2 cells (CD19+CD5−B220hi) as well as for VH12/Vκ4 transgenic wild-type and DKO CD19+ B cells. One representative result of three independent experiments is shown. (e) Frequency of VH12− and VH12+ PtC liposome-binding cells (gated as shown in Fig. 6e) of peritoneal wild-type and Cd19−/− IgM+CD5+ B-1a cells. Horizontal bars indicate mean value, and error bars represent s.d. NS P > 0.05, * P < 0.05 as determined by the Student’s t-test. The data of eight wild-type and seven Cd19−/− mice analyzed in three independent experiments are shown.
