|
|
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<.01). (B) Reduced cellularity was consistently observed in Cited2−/− fetal liver at 15.5 dpc (n=11) compared to Cited2+/+ controls (n=13; P<.01). Error bars are SEM.
- 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<.01; average ± SD).
- 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.
|
|
