|
|
Blood, Vol. 112, Issue 12, 4485-4493, December 1, 2008
FoxO3a regulates hematopoietic homeostasis through a negative feedback pathway in conditions of stress or aging
Blood Miyamoto et al.
112: 4485
Supplemental materials for: Miyamoto et al
Files in this Data Supplement:
- Figure S1. Aged FoxO3a-deficient mice develop neutrophilia (JPG, 63.9 KB)
-
(A) Peripheral blood counts of aged (15–18 months old) FoxO3a+/+ or FoxO3a−∕− mice. WBCs, white blood cells; Hb, hemoglobin. Data are mean counts (×1,000/µl) ± s.d. of WBCs, (g/dl) ± s.d. of Hb and (×1,000/µl) ± s.d. of platelets in aged FoxO3a-deficient (black columns; n = 5) or wild-type (white columns; n = 5) mice. *P < 0.01. (B) Images of peripheral blood isolated from 8 to 12-week-old or 15 to 18-month-old FoxO3a+/+ or FoxO3a−∕− mice and stained with May-Gruenwald Giemsa. Bar=100 µm. (C) Peripheral blood isolated from 8-week or 18-month-old FoxO3a-deficient or wild-type mice were stained with May-Gruenwald Giemsa, and the frequency of neutrophils was scored. Data are mean frequency (%) ± s.d. of neutrophils in peripheral WBCs of FoxO3a+/+ mice (white columns; n = 5) or FoxO3a−∕− (black columns; n = 5). *P < 0.01, NS; not significant.
- Figure S2. Normal recovery of platelets and hemoglobin in FoxO3a-deficient mice following myelo-suppression (JPG, 41.6 KB)
-
Platelet and hemoglobin (Hb) counts after 5-FU injection. Data are mean counts (×1,000/µl) ± s.d. of platelets and (g/dl) ± s.d. of Hb in wild-type (white squares; n = 5) FoxO3a-deficient (black squares; n = 5) mice.
- Figure S3. Comparable concentrations of serum G-CSF in FoxO3a-deficient and wild-type mice before and after 5-FU treatment (JPG, 37.4 KB)
-
Serum G-CSF concentrations in FoxO3a-deficient (black column; n = 5) and wild-type (white column; n = 5) mice before (A) and on day 18 after (B) 5-FU injection. Data are mean ± s.d. of the concentration of serum G-CSF (pg/ml, NS; not significant).
- Figure S4. FoxO3a-deficient cells are hyper-responsive to G-CSF (JPG, 23 KB)
-
BM mononuclear cells were isolated from 8 to 12-week-old FoxO3a-deficient or wild-type mice, and cultured in the presence of indicated concentrations of G-CSF in methylcellulose medium. After 7 days of culture, the number of colonies was counted. Data are mean ± s.d. of the number of colonies derived from FoxO3a-deficient (black columns; n = 5) and wild-type cells (white columns; n = 5) in the presence of indicated concentrations of G-CSF. *P<0.01.
- Figure S5. Negative regulation of granulopoiesis by FoxO3a over-expression (JPG, 52.1 KB)
-
BM mononuclear cells were isolated from 8 to 12-week-old wild-type mice, infected with FoxO3a-EGFP- or EGFP-expressing retrovirus and cultured in the presence of 10 ng/ml G-CSF or GM-CSF. After 7 days of culture, the frequency of Gr1-positive cells (%) in PI−EGFP+ cells was analyzed by FACS.
- Figure S6. ROS-independent over-stimulation of granulopoiesis in FoxO3a-deficient mice under myelo-suppressive stress (JPG, 71.4 KB)
-
(A) Experimental procedure. (B) Data are means ± s.d. of WBC counts (×1,000/µl) following 5-FU injection with or without NAC treatment. White circles, WBC counts in wild-type mice without NAC (n = 5); black squares, WBC counts in FoxO3a-deficient mice without NAC (n = 5); black triangles, WBC counts in FoxO3a-deficient mice with NAC (n = 5). (C) Frequency of neutrophils in peripheral blood WBC following 5-FU injection in FoxO3a-deficient (white columns; n = 5) or wild-type (black columns; n = 5) mice under treatment with or without NAC. Data are mean frequency (%) ± s.d. of neutrophils in peripheral WBCs. *P < 0.01; NS, not significant. (D) Comparable intracellular ROS levels in HSCs of FoxO3a-deficient (black columns; n = 3) and wild-type mice (white columns; n = 3) 2, 10 and 17 days after 5-FU injection. Data are mean fluorescence intensity ± s.d. of DCF-DA in HSCs. *P < 0.01; NS, not significant. (E) FoxO3a-deficient HSC function was determined by long-term culture, LTC-IC assays. HSCs were isolated from FoxO3a-deficient or wild-type mice, and pre-cultured on OP9 stromal cells in the presence or absence of 100 µM NAC for 2 or 6 weeks. Then cells were harvested, cultured in methylcellulose medium for 7 days and the number of colonies was counted. Data are mean ± s.d. of the number of colonies. *P<0.05. **P<0.01.
- Figure S7. Spred2 but not SOCS3 is downregulated in FoxO3a-deficient cells (JPG, 36.9 KB)
-
Data are mean ± s.d. of expression Spred2 or SOCS3I expression relative to GAPDH in FoxO3a-deficient LSK cells (black columns; n = 3) relative to wild-type LSK cells (white columns; n = 3) transplanted into recipient mice. *P < 0.01, NS; not significant.
- Figure S8. Upregulation of p27Kip1 is inhibited in HSCs from FoxO3a-deficient mice after 5-FU treatment (JPG, 116 KB)
-
(A) p27Kip1 immunostaining in BM mononuclear cells (unfractionated) or Lin−Sca-1+ cells 2 days after 5-FU injection in 8 to 12-week-old FoxO3a-deficient or wild-type mice. Bar=100 µm. Data are means ± s.d. of p27Kip1 expression relative to GAPDH in FoxO3a-deficient Lin−Sca-1+ or unfractionated cells (black columns; n = 3) relative to wild-type unfractionated cells (white columns; n = 3). (B) p21Cip1 immunostaining in BM mononuclear cells (unfractionated) or Lin−Sca-1+ cells 2 days after 5-FU injection in 8 to 12-week-old FoxO3a-deficient or wild-type mice. Bar=100 µm. Data are means ± s.d. of p21Cip1 expression relative to GAPDH in FoxO3a-deficient Lin−Sca-1+ or unfractionated cells (black columns; n = 3) relative to wild-type unfractionated cells (white columns; n = 3). (C) p57Kip2 immunostaining in BM mononuclear cells (unfractionated) or Lin−Sca-1+ cells 2 days after 5-FU injection in 8 to 12-week-old FoxO3a-deficient or wild-type mice. Bar=100 µm. Data are means ± s.d. of p57Kip2 expression relative to GAPDH in FoxO3a-deficient Lin−Sca-1+ or unfractionated cells (black columns; n = 3) relative to wild-type unfractionated cells (white columns; n = 3).
- Figure S9. Aged mice develop over-increase of WBCs (JPG, 36.4 KB)
-
Peripheral WBC counts of aged (12–18 months old) and younger (<12-week-old) mice during hematopoietic recovery after 5-FU treatment. WBCs, white blood cells.
|
|
