a TMRM FI (Median) b TMRM FI (Median) c 20 15 10 5 0 8 6 4 2 0 pro-b large pre-b small pre-b 0 10 20 30 40 50 60 70 80 90 100 TMRM (nm) pro-b large pre-b small pre-b 0 1 2 4 8 16 32 64 128 256 CCCP (mm) CCCP (µm) Rag1 -/- Rag1 -/- ;33.C9HCki CCCP (100 µm) TMRM CD19 TMRM CD19 Supplementary Figure S1 Calibration of specific TMRM fluorescence A) BM of WT mice was stained with increasing concentrations of TMRM and gated on living, single pro B (CD19 +, ckit + ), large pre B (CD19 +, CD25 +, FSC Int high ) and small pre B cells (CD19 +, CD25 +, FSC Int low ). Further experiments were performed with a TMRM concentration in the linear range (25 nm). B) BM of WT mice was stained with 25nM of TMRM with increasing concentrations of Carbonyl cyanide 3- chlorophenlyhydrazon (CCCP) and gated on living, single pro B (CD19 +, ckit + ), large pre B (CD19 +, CD25 +, FSC Int high ) and small pre B cells (CD19 +, CD25 +, FSC Int low ). Further experiments were performed with a CCCP concentration that blocked TMRM fluorescence completely (100µM). C) Pro and pre B cells from the BM of Rag1 -/- or Rag1 -/- ;33.C9µHCki mice were positively sorted by anti CD19 MACS, attached to glass slides, stained with TMRM (100nM) and anti CD19 antibody coupled to FITC in the absence or presence of CCCP (100µM) and living cells were analyzed by confocal microscopy. Scale bar, 12µm.
Supplementary Figure S2 Homing of EFhd1tg and WT B cells into lymphoid organs A) Injection mix of ef670 and CSFE labelled EFhd1tg and WT splenic B cells in PBS isolated by MACS CD43 depletion (B) Gating strategy to detect homing of EFhd1tg and WT B cells into BM, spleen and inguinal LN (iln). Splenic B cells from EFhd1tg and WT mice were isolated, enriched by MACS CD43 depletion (Milteny) and stained either with CSFE or ef670. Equal cell numbers of WT CSFE or ef670 labelled cells were mixed with ef670 or CSFE labelled EFhd1tg cells, respectively.c57bl/6 mice were injected with 2x10 7 mixed cells and homing into BM, spleen and iln were analyzed after 3 or 24 hours p.i. by flow cytometry by gating on singlet living lymphocytes and CSFE/eF670 positive cells. (C) The ratio of WT/ EFhd1tg B cells found in BM, spleen and iln is represented as mean+ SEM of 8 mice per time point from 2 experiments. 4 mice were analyzed with WT CSFE/EFhd1tg ef670 labelled cells and 4 with WT ef670/ EFhd1 CSFE labelling to exclude an effect of either dye treatment.
Supplementary Figure S3 Cellular composition of EFhd1 tg and WT pro B cell ex vivo IL-7 cultures after 1 week. A) Sorted pro B cells were cultured with 5 ng/ml IL-7 as described previously. After 7-8 days cells were stained for surface CD19, ckit, CD25 and IgM expression. Freshly isolated C57BL/6 BM was included as reference. The stained cells were then analyzed by flow cytometry after gating on singlet and living lymphocytes. Representative plots of one of 3 independent cultures are shown (B) Pro and pre B cells were further discriminated by size. CD19 +, sigm - cells were analyzed for c-kit and CD25 expression. In WT BM cells CD25 was used as positive marker for pre B cells. In Il-7 culture, the pre B cells derived from differentiated pro B cells were defined as CD19 +, ckit -, sigm -. The cell size of pro and pre B cells was compared by overlay of the FS INT histogram.
Supplementary Figure S4 Proliferation of EFhd1 tg and WT pro B cells in IL-7 cultures A) Sorted pro B cells were seeded at 1x10 5 cells/ml and cultured in OptiMEM10 with indicated IL-7 concentrations. IL-7 was replaced every 3 days, new medium added on day 4 and the cells were split to original density on day 5 and 8. Viable cells in the culture were counted with a Neubauer chamber, n=3-4 independent cultures of sorted pro B cells from pooled BM of 3 mice per genotype. Values are represented as mean+ SEM (B) Sorted pro B cells were stained with ef670 and uniform staining on day 0 assed by flow cytometry. The ef670 staining was then analyzed after 3 days in culture with 5 ng/ml IL-7. Cell proliferation was analyzed by automated gating using the FlowJo Proliferation tool. Bar diagrams show the percentage of cells in culture that have divided during the 3 day culture and the mean division number/dividing cell as assed by FlowJo. The data is represented as mean+sem of 3 independent sorts with pro B cells of 3 pooled mice per genotype.
Supplementary Figure S5 Ex vivo differentiation of EFhd1tg and WT pro B cells in an IL-7 culture A) Sorted pro B cells (CD19 +, B220 +, ckit + ) were seeded at 1 x 10 5 cells/ml and cultured in OptiMEM10 with indicated IL-7 concentrations. The IL-7 was replaced every 3 days, new medium added on day 4 and the cells were split to original density on day 5. On day 3 and 6 cells were stained with fluorescently labelled antibodies against CD2 and IgM and analyzed by flow cytometry. After gating on living lymphocytes a quadrant plot allows the evaluation of pro B cell differentiation in culture as pro B/large pre B cells are double negative while small pre B cells gain expression of CD2 and immature B cells express CD2 as well a sigm +. Representative FACs plots after gating on living lymphocytes are shown for day 3 and day 6 in culture with 0, 0.5 and 5 ng/ ml IL-7 (B) Pro-B cell proliferation/differentiation was analyzed on day 3 and 6 for cells cultured in the presence of 0, 0.5 and 5 ng/ml IL-7. The proportion of cells in the different developmental stages are represented as mean+ SEM, n=3 independent pro B cell sorts of pooled BM from 3 EFhd1tg or WT mice.