Display Settings:

Items per page

Results: 7

1.
Figure 5

Figure 5. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

Proliferation potential of L3MBTL1-KD human hematopoietic progenitor cells. (A) The maintenance of CD34 expression was evaluated in L3MBTL1-KD CB cells by flow cytometry. GFP+CD34+ CB cells were cultured with SCF, FLT-3, IL-6, and TPO. *P < .005. (B) Cell counts of L3MBTL1-KD CB cells were monitored at different time points in liquid culture with SCF, FLT-3, IL-6, and TPO. (C) Seventy-two hours after lentiviral infection, sorted GFP+CD34+ HPCs were placed in CFU assays and the number of CFUs quantified. (D) p57 mRNA expression was assessed by quantitative RT-PCR in L3MBTL1-KD HPCs, with and without exposure to 200pM TGF-β1 for 2 hours (n = 2).

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.
2.
Figure 7

Figure 7. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

L3MBTL1 restricts erythroid differentiation. (A) GlyA expression was assessed by FACS in the L3MBTL1-HA expressing CMK, U937, HEL, and K562 cells compared with the MIGR1 (empty vector) transduced cells. (B) p16 protein expression levels were determined by Western blot analysis in L3MBTL1-HA HEL cells versus the MIGR1 (empty vector) control HEL cells. JNK and L3MBTL1 levels are also shown. Tubulin served as the loading control. (C) GlyA expression in L3MBTL1-HA expressing CD34+ cells after 3 days of culture with SCF 100 ng/mL and Epo 6 IU/mL, compared with the MIGR1 (empty vector) transduced cells. (D) L3MBTL1 mRNA expression levels in retrovirally infected CD34+ cells, quantified by quantitative PCR (n = 3).

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.
3.
Figure 3

Figure 3. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

Knockdown of L3MBTL1 induces further erythroid differentiation of K562 erythroleukemia cells. (A) K562 cells, grown in RPMI medium supplemented with 10% fetal bovine serum, without exogenous cytokines or hemin, were infected with lentiviral constructs targeting luciferase (shLUC) or L3MBTL1 (sh1 and sh2). GFP+ cells, sorted by FACS at 72 hours after infection, were analyzed for GlyA expression by flow cytometry. (B) GFP+ K562 cells, before and after exposure to hemin (50μM) for 4 days, were stained with benzidine to assess their Hb content. (C) K562 cells were treated with 50μM hemin for 4 days and the L3MBTL1 mRNA level assessed in the hemin-exposed versus the nontreated cells by quantitative RT-PCR (n = 3).

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.
4.
Figure 4

Figure 4. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

L3MBTL1-KD leads to expansion of erythroid progenitors in long-term culture. (A) Sorted 4 × 105 GFP+ CD34+ were plated on MS5 stromal cell layer and cultured for 5 weeks. At week 5, the colonies were examined using an inverted optical microscope. The red arrows indicate the cobblestone area forming cells in the wt and shLUC figures. The red arrows indicate the overgrowth of progenitor cells (predominantly erythroid) in the sh1 and sh2 photographs. (B) The expression of GlyA on floating cells from 5-week LTC-IC cultures was evaluated by flow cytometry. (C) CAFC colony numbers were evaluated at week 5 of MS-5 stromal cell–based culture (n = 2). (D) Week 5 LTC-IC cells were plated on methylcellulose, and the secondary BFU-E colonies were scored after 10 days. The ratio of BFU-E colonies is shown, based on BFU-E numbers in the control cells.

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.
5.
Figure 2

Figure 2. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

L3MBTL1 knockdown specifically promotes erythroid, but not myeloid or megakaryocytic, differentiation of human CD34+ cells. (A) L3MBTL1 expression levels were assessed by quantitative RT-PCR in normal CD34+ CB cells. The cells were placed in different culture conditions stimulating erythroid, myeloid, and megakaryocytic differentiation for 2, 5, and 7 days. Total RNA was extracted from 2 × 105 cells. (B) Seventy-two hours after lentiviral infection, GFP+CD34+ cells were cultured in myeloid conditions for 7, 9, and 11 days, and the expression of myeloid-specific markers CD11b (CD14 and CD33 not shown) was assessed by flow cytometric analysis (n = 3). (C) Seventy-two hours after lentiviral vector infection, the GFP+CD34+ cells were cultured in megakaryocytic conditions for 7, 9, and 11 days, and expression of the megakaryocytic-specific marker CD41 was assessed by flow cytometric analysis (n = 3). (D) GFP+CD34+ cells, 72 hours after lentiviral vector infection, were cultured in erythroid conditions for 7, 9, and 11 days, and expression of the erythroid-specific marker GlyA was assessed by flow cytometric analysis (n = 3).

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.
6.
Figure 1

Figure 1. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

Knockdown of L3MBTL1 promotes the erythroid differentiation of human hematopoietic CD34+ progenitor cells. (A) Lentiviral constructs expressing shRNAs targeting luciferase (control) or L3MBTL1 (sh1 and sh2) led to efficient knockdown in primary CB CD34+ cells, as assessed by Western blot and quantitative RT-PCR. shRNAs of a different backbone (empty vector and sh3) also yielded efficient knockdown of L3MBTL1. All the presented data were confirmed using both sets of constructs. (B) Expression of CD71 and GlyA on human HSPCs, as assayed by flow cytometric analysis at days 7, 9, and 11 of Epo-induced culture. (C) Expression of CD71 and GlyA on human HSPCs, cultured with different concentrations of Epo, as assayed by flow cytometric analysis at 7 days. (D) Cells from panel B were stained with May-Grunwald-Giemsa on day 7 of Epo-induced culture, and their morphology was captured by light microscopy. (E) Epo-exposed cells at days 7, 9, and 11 of Epo-induced culture were resuspended in benzidine solution, and cells that stained dark blue-green were scored as positive.

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.
7.
Figure 6

Figure 6. From: Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera.

Primary hematopoietic cells lacking L3MBTL1 show Epo-independent phosphorylation of STAT5, AKT, and MAP kinase. (A) Primary GFP+ CD34+ cells were harvested after one week in erythroid culture and lysed, according to standard protocols. Cultured CD34+ cells from MPN patients bearing the JAK2V617F mutation were harvested after 1 week of culture in Epo. Phosphorylation of STAT5, AKT, and MAPK was assessed by Western blot. The protein levels of JAK2, STAT5, AKT, MAPK, and L3MBTL1 were also assessed. (B) Primary GFP+ CD34+ cells were harvested after one week in erythroid culture and FOXO phosphorylation, and JNK expression levels were assessed by Western blot assay. Tubulin serves as a loading control for panels A, B, and E. (C) A total of 2 × 107 GFP+ CD34+ cells, plated in erythroid culture conditions, were tested for Ras activation, by detection of GTP-bound Ras. Cells were lysed and incubated with (GST)-Raf-RBD fusion protein coupled to glutathione agarose beads. GTP-bound Ras was detected by Western blotting using an isoform-specific antibody. (D) Expression of the erythroid markers CD71 and GlyA was evaluated on L3MBTL1-KD CB cells cultured without Epo, in SCF, FLT-3, IL-6, and TPO. (E) After 1 week in culture without Epo, the GFP+ CD34+ cells were lysed, and phosphorylation of STAT5 and AKT was assessed by Western blot analysis. The protein levels of JAK2, STAT5, AKT, MAPK, Raf-1, p16, and L3MBTL1 were also assessed.

Fabiana Perna, et al. Blood. 2010 October 14;116(15):2812-2821.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk