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1.
Fig. 6

Fig. 6. CALM-AF10 interaction with clathrin does not influence global H3K79 methylation. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

(A) Nuclear extracts, prepared from 293T cells transfected with CALM-AF10 constructs or leukemia cells from individual transplanted CALM-AF10+ mice, were immunoblotted for H3K79-2me and histone H3. Quantification of H3K79-2me Western blot immunostaining, normalized to H3, confirms a decrease in H3K79 methylation upon CALM2091AF10 expression compared to GFP control in 293T and mouse cells. Global hypomethylation after CALM1926AF10 expression is less pronounced. (B) Nuclear extracts were prepared from the CALM2091AF10+ CA2091-CL1 cell line, transfected with non-targeting (NT), CALM-AF10 (hAF10) or clathrin specific siRNA pools (CLTC-pool1, pool2). A representative blot indicates H3K79 methylation is increased after knock-down of CALM-AF10, but not clathrin (~92% knockdown for CLTC-pool1). Quantification of the average H3K79-2me, clathrin and CALM2091AF10 protein levels (Right panel, 3 independent experiments).

Angela Stoddart, et al. Oncogene. ;31(4):494-506.
2.
Fig. 7

Fig. 7. Self-association properties of CALM2091AF10 visualized by acceptor and donor photobleaching FRET. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

(A) 293T cells were transfected with chimeric transcripts of CALM2091AF10 fused to YFP and CFP (CA2091-YFP + CA2091-CFP). A representative image of YFP and CFP fluorescence before and after photobleaching and corresponding FRET efficiency is shown. The percent of CALM2091AF10 aggregates displaying FRET and the average FRET efficiency for bleached regions (n=147) and control (non-bleached) regions (n= 97) ± the standard error of the mean, for three individual experiments are shown. (B) 293T cells were transfected with CA2091-CFP + CA2091-YFP, CA1926-CFP + CA1926-YFP, CA2091-CFP or CA1926-CFP. Cells were bleached using the donor excitation wavelength of 458 nm, a time-lapse of images was captured, and a fluorescence decay constant was measured. Mean decay constants in cells expressing both CA2091-CFP and CA2091-YFP (n=110) have a slower decay constant than CA2091-CFP alone (n=88) (P<0.0001), suggesting the occurrence of FRET.

Angela Stoddart, et al. Oncogene. ;31(4):494-506.
3.
Figure 5

Figure 5. CALM2091AF10 localizes to the nucleus and does not influence Kit internalization in a myeloid line derived from CALM2091AF10+ AML cells. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

(A) Protein expression of CALM2091AF10, detected using an anti-FLAG antibody, 48 hours after nucleofection with siRNA specific for human AF10 (hAF10) or non-targeting (NT) control siRNA in mouse CA2091-CL1 cells, an IL-3 dependent line derived from CALM-AF10+ AML mouse cells. (B) CA2091-CL1 cells were transfected with siRNAs: NT-non-targeting; hAF10-specific for CALM-AF10. Cells were treated with 200ng/ml SCF for times indicated, and KIT receptor remaining on the cell surface was measured. Fetal liver cells are shown as a positive control for KIT internalization. Knock-down of CALM-AF10 expression (hAF10 siRNA) does not increase KIT internalization. (C) Localization of CALM2091AF10, expressed in CA2091-CL1 cells, during interphase (1) and metaphase (2,3). Original magnification, 787.5X.

Angela Stoddart, et al. Oncogene. ;31(4):494-506.
4.
Fig. 2

Fig. 2. The absence of the clathrin-binding region of CALM alters the phenotype of CALM-AF10+ myeloid neoplasms. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

(A) Schematic representation of CALM, AF10 and CALM-AF10 fusion proteins. The two breakpoints in CALM, and one of the four breakpoints in AF10 are indicated by black lines and the base pair position. The two CALM-AF10 fusion proteins used in this study are shown. (B) Survival curves of mice injected with progenitor cells transduced with CALM2091AF10, CALM1926AF10 or MIGR1 (control vector). CA= CALM-AF10; 2ND = secondary recipients of primary MPD or AML cells. (C) Fluorescence-activated cell sorter analysis of cells isolated from spleen of CALM-AF10 mice. Analysis of GFP-gated cells reveals an enrichment of myeloid lineage-specific markers (CD11b+Gr-1med-hi) and decrease in erythroid cells (CD71+Ter119+) in CALM2091AF10 and CALM1926AF10 mice compared to control (MIGR1) mice. (D) Peripheral blood, bone marrow aspirates and spleen touch preparations (TP) were stained with Wright-Giemsa. Paraffin-embedded liver and spleen sections were stained with hematoxylin and eosin. Original magnification ×100 for spleen and liver; ×500 for blood, spleen-TP and bone marrow; ×1000 for spleen and insets.

Angela Stoddart, et al. Oncogene. ;31(4):494-506.
5.
Fig. 4

Fig. 4. Internalization and proliferation is not altered in CALM-AF10+ hematopoietic or leukemia cells. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

Internalization of KIT (A), CXCR4 (B), or TF (C) receptor, gated on GFP+ bone marrow cells isolated from CALM2091AF10+ AML mice (solid lines) or CALM1926AF10+ MPD mice (dashed lines), compared to control MIGR1 mice. (D) Growth curve of trypan blue-excluded viable BaF3 cells expressing CALM-AF10 or control MIGR1 vector grown in low (0.01 ng/ml, dashed line) or high (1ng/ml, solid line) levels of IL-3,. (E) BaF3 cells expressing CALM-AF10 or control MIGR1 vector were serum starved, treated with IL-3 for times indicated, and lysates were immunoblotted with a phospho-Erk 1/2 antibody. No increase in Erk phosphorylation, normalized to Erk 2 levels, was observed in CALM-AF10 expressing cells. (F) To estimate the frequency of tetraploid metaphase cells, BaF3 cells were treated with Colcemid™, and metaphase cells were prepared with standard cytogenetic techniques. A higher percent of tetraploid metaphase cells were found in BaF3 cells expressing CALM-AF10 compared to MIGR1 controls. An average of three independent experiments is shown. In each experiment, 30-50 metaphases were scored for each line.

Angela Stoddart, et al. Oncogene. ;31(4):494-506.
6.
Fig. 3

Fig. 3. Differential gene expression in CALM2091AF10 AMLs and CALM1926AF10 MPDs. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

(A) RNA expression levels were quantified by real-time RT-PCR. Fold change in RNA expression in spleen cells from mice with AML relative to MPD is shown. A positive value indicates the gene is up-regulated in AML, and a negative value indicates it is down-regulated in AML compared to MPD samples. (B) Hoxa cluster gene expression relative to MIGR1 control (normalized to 1). Whereas Hoxa genes were significantly up-regulated compared to controls, there was no difference between AML and MPD samples. Results are expressed as the mean ± standard error from three independent experiments. (C) Fetal liver (FL) progenitors, infected with retroviral vectors expressing either CALM2091AF10 or CALM1926AF10 were grown in vitro for 10-12 days; changes in gene expression relative to the MIGR1 control were examined by real-time PCR (four independent experiments). To assess differences between CALM-AF10 constructs, relative gene expression was set as ‘1’ for CALM2091AF10+ cells. Hoxa5, Hoxa7, Hoxa9, Meis1, and Bmi1 gene expression was consistently increased ~2 fold in CALM1926AF10+ cells vs. CALM2091AF10+ cells (P<0.01).

Angela Stoddart, et al. Oncogene. ;31(4):494-506.
7.
Fig. 1

Fig. 1. CALM-AF10 inhibits clathrin-mediated endocytosis in 293T cells. From: The Clathrin-Binding Domain of CALM-AF10 Alters the Phenotype of Myeloid Neoplasms in Mice.

(A) Schematic diagram of the CALM protein indicating nucleotide positions where fusion to AF10 occurs. The large black box refers to the ANTH domain (PIP2-binding) and the small box refers to the location of exon 13, which is alternatively spliced and contains a DPF peptide motif involved in, but not essential for, AP2 binding. Clathrin-coated vesicle fractions (CCV) or total cell lysates, isolated from NIH3T3 cells infected with retroviral vectors expressing FLAG-tagged CALM constructs ending at breakpoint 1926 or 2091, with or without exon 13, were resolved by electrophoresis and blotted with FLAG, clathrin and actin-specific antibodies. (B, C) 293T cells were transfected with chimeric transcripts of CALM2091, CALM1926, CALM2091AF10 or CALM1926AF10 fused to GFP. Expressed proteins were detected by GFP fluorescence and nuclei were visualized by DAPI staining. To block nuclear export, transfected cells were treated with 20 nM leptomycin B, where indicated. Original magnification × 787.5 for all panels. (D) Forty-eight hours after transfection with GFP-fused constructs, a total of 150 GFP+ 293T cells were scored for uptake of fluorescent transferrin, from three independent experiments (50 cells per experiment). There was a significant difference in transferrin uptake between control (EGFP-N1) and CALM1926AF10 (P<0.02) and control and CALM2091AF10 (P<0.0001), with and without exon 13.

Angela Stoddart, et al. Oncogene. ;31(4):494-506.

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