Cited2 is required for normal hematopoiesis in the murine fetal liver -- Chen et al. 110 (8): 2889 Data Supplement -

Supplemental materials for: Chen et al, Vol. 110, Issue 8, 2889-2898. -- Blood

Blood, Vol. 110, Issue 8, 2889-2898, October 15, 2007

Cited2 is required for normal hematopoiesis in the murine fetal liver
Blood Chen et al. 110: 2889

Supplemental materials for: Chen et al, Vol. 110, Issue 8, 2889-2898.

Files in this Data Supplement:

Document 1. Supplemental materials and methods (PDF, 90.7 KB)
Figure S1. Decreased fetal liver cellularity in Cited2^{−/−} embryos at 14.5 dpc and 15.5 dpc (JPG, 32.3 KB). -
Fetal livers from 14.5 dpc and 15.5 dpc were harvested and single cell suspension was prepared by passing through a 1-mL pipette and filtered through 100-µm cell strainer. Cell number was counted using a hemocytometer. (A) At 14.5 dpc, Cited2^{−/−} fetal liver (n=6) displayed significantly decreased cellularity compared to Cited2^+/+ controls (n=7; P−/− fetal liver at 15.5 dpc (n=11) compared to Cited2^+/+ controls (n=13; P
Figure S2. Decreased frequency of CD45⁺ LSK cells in Cited2^{−/−} fetal liver (JPG, 131 KB). -
(A) Fetal liver cells from Cited2^{−/−} (n=3) and Cited2^+/+ littermate control (n=4) at 14.5 dpc were stained with PE-conjugated lineage markers, PerCP-Cy5.5-conjugated CD45.2, APC-conjugated-c-Kit, and FITC-conjugated Sca-1 antibodies, and the percentage of CD45⁺ LSK cells were analyzed as shown by the representative lineage gates. (B) The frequency of CD45⁺ LSK was compared between Cited2^{−/−} and Cited2^+/+ littermate control and revealed significant reduction in Cited2^{−/−} fetal liver (P
Figure S3. Decreased CFU frequency of hematopoietic progenitor cells in Cited2^{−/−} fetal liver (JPG, 45.5 KB). -
Fetal liver cells (2 × 10⁴) from each sample were plated in triplicate cultures of methylcellulose-based medium supplemented with 3 units/mL Epo, 10 ng/mL mouse recombinant IL-3, 10 ng/mL human recombinant IL-6, and 50 ng/mL mouse recombinant stem cell factor. The number of BFU-E, CFU-GM, and CFU-GEMM was determined after 7 to 12 days of culture. (A) At 13.5 dpc, the number of BFU-E, CFU-GM, and CFU-GEMM was significantly reduced in Cited2^{−/−} fetal livers (n=5) compared to the wild type littermate controls (n=5). (B) At 14.5 dpc, decreased numbers of BFU-E, CFU-GM, and CFU-GEMM were observed in Cited2^{−/−} fetal livers (n=4) compared to the wild type littermate controls (n=3). (C) Decreased numbers of BFU-E, CFU-GM, and CFU-GEMM were observed in Cited2^{−/−} fetal liver (n=4) compared to the wild type littermate control (n=6) at 15.5 dpc. In addition, Cited2^+/− fetal livers also showed decreased colony formation at corresponding stages (n=4 at 13.5 dpc, n=5 at 14.5 dpc, and n=4 at 15.5 dpc.). Error bars are SEM.
Figure S4. Chimerism analysis in mice with Cited2^+/+ and Cited2^{−/−} primary and secondary transplants (JPG, 53.7 KB). -
Chimerism analysis showed high donor chimerism in multiple lineages in mice with Cited2^+/+ and Cited2^{−/−} primary transplants (A) and significantly reduced donor chimerism in mice with Cited2^{−/−} (n=3) versus Cited2^+/+ (n=3) secondary transplants (B). Error bars are SEM.