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First published online 21 March 2006
doi: 10.1242/jcs.02870

Journal of Cell Science 119, 1483-1493 (2006)
Published by The Company of Biologists 2006
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The transcription factor B-Myb is essential for S-phase progression and genomic stability in diploid and polyploid megakaryocytes

Paloma García and Jon Frampton*

Institute for Biomedical Research, Birmingham University Medical School, Edgbaston, Birmingham, B15 2TT, UK


Figure 1
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  		Fig. 1. B-Myb is expressed in endoreplicating cells during megakaryocytic differentiation. Cells were cultured from an initial density of1.5x105/ml in the absence or presence of 10–8 M TPA for 24, 48 or 72 hours. (A) Propidium iodide staining of TPA-treated and untreated HEL, CMK and U937 cells. The vertical axis indicates the relative number of cells and the horizontal axis indicates the relative red fluorescence (FL2) on a logarithmic scale as a measure of DNA content. The positions of peaks representing cells with DNA content of 2, 4, 8 and 16C are indicated. (B) 50 µg of total protein extract from cells exponentially growing (0) or treated with TPA for 24, 48 and 72 hours were subjected to SDS-PAGE, transfered to a membrane and probed with antibodies against B-Myb. Coomassie Blue staining was performed with the upper part of the gel as loading control. (C) Flow cytometric analysis of HEL cells that were either exponentially growing (0) or treated with 10–8 M TPA for 24 and 48 hours. Expression of B-Myb (lower panels) or IgG control (upper panels) was detected by indirect immunofluorescence using anti-IgG or anti-B-Myb in conjunction with FITC-conjugated goat anti-rabbit IgG (FL1, vertical axis, linear scale), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis, linear scale). In the right panel is shown an enlargement of the profile for HEL cells treated with TPA for 48 hours, the arrow indicating cells with active endoreplicating S phase. (D) Flow cytometric analysis of B-Myb expression in HEL cells treated with 10–8 M TPA for 48 hours as described in (C) except that blocking peptide was included in the sample in the lower panel. (E) HEL, CMK and U937 cells were cultured from an initial density of 1.5x105/ml in the absence or presence of 10–8 M TPA for 24, 48 or 72 hours. cDNA was prepared from 3 µg RNA and semi-quantitative PCR analysis was performed to measure the relative expression of B-myb. HPRT was used to standardise loading of equal amounts of cDNA. The upper and lower panels show the RT-PCR analysis of HPRT and B-myb RNA, respectively. The size of PCR products was 620 bp for HPRT and 310 bp for B-myb. The PCR reactions were sampled at cycles 25, 28, 31, 34 and 37 for reactions using HEL cDNA and at cycles 22, 25, 28, 31 and 34 for reactions using CMK and U937 cDNA.

 

Figure 2
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  		Fig. 2. B-Myb protein is expressed in primary mature megakaryocytes. (A) E15 foetal liver cells were cultured in vitro for 4 days in the presence of TPO, then immunodepleted and subjected to fractionation on a discontinuous BSA gradient. (B) Propidium iodide staining of cells from Fractions 1 and 4. The vertical axis indicates the relative number of cells and the horizontal axis indicates the relative red fluorescence (FL2) on a logarithmic scale as a measure of DNA content. Peaks representing each ploidy class are labelled. (C) Megakaryocytes from Fractions 1 and 4 were cytospun and stained for acetylcholinesterase. (D) 50 µg of total protein extract from cells in Fractions 1 and 4 were subjected to SDS-PAGE and detected by western blot with antibody against B-Myb. The position of relevant molecular weight standards is shown on the left side of the blot. Coomassie Blue staining was performed with the upper part of the gel as a loading control. (E) Expression of B-Myb was detected in E15 foetal liver (FL) cells cultured in the presence of TPO for 4 days, by indirect immunofluorescence using a B-Myb polyclonal antibody or IgG control and FITC-conjugated goat anti-rabbit IgG (FL1, vertical axis, logarithmic scale), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis, logarithmic scale). The polygon shows the position of cells stained in parallel with the isotype control. (F) DNA synthesis in mature megakaryocytes. Cells that had differentiated for 5 days were labelled with BrdU for 16 hours. Incorporation of BrdU was detected by indirect immunofluorescence using a mouse anti-BrdU antibody and PE-conjugated goat anti-mouse Ig secondary antibody (red). Nuclei were stained with DAPI (blue). The upper panels are of a mature megakaryocyte that had not been labelled with BrdU. The middle and lower panels are examples of cells that have incorporated BrdU, representing, respectively, a mature polyploidy megakaryocyte and a terminally differentiated, platelet-producing cell.

 

Figure 3
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  		Fig. 3. Overexpression of B-Myb influences endoreplication in megakaryoblastic cells. (A) Diagram of the pIRES-EGFP and B-Myb-IRES-EGFP plasmids. HEL cells were transfected with pIRES-EGFP control plasmid or B-Myb-IRES-EGFP and sorted for GFP expression after 18 hours. CMV Pr, cytomegalovirus promoter. (B) 50 µg of total protein from GFP+ and GFP– populations were subjected to SDS-PAGE and detected by western blot with antibodies against B-Myb and ß-actin. (C) Cell-cycle distribution of sorted GFP– cells and GFP+ cells treated with TPA for 72 hours; the middle and right panels correspond to GFP+ cells from transfections with the control vector and B-Myb-IRES-EGFP, respectively. The vertical axis indicates the relative number of cells and the horizontal axis indicates the relative red fluorescence (FL2) on a logarithmic scale as a measure of DNA content. (D) Flow cytometric analysis of GFP+ cells that were in the presence of TPA for 18 hours, followed by 3 hours in the presence of TPA and BrdU. Incorporation of BrdU was detected by indirect immunofluorescence using a FITC-conjugated mouse anti-BrdU monoclonal antibody and FITC-conjugated mouse IgG1 isotype control (FL1, vertical axis, logarithmic scale), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis, linear scale). The left and right panels correspond to GFP+ cells from transfections with the control vector and B-Myb-IRES-EGFP, respectively.

 

Figure 4
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  		Fig. 4. Downregulation of B-Myb by RNA interference. HEL cells treated with 10–8 M TPA for 16 hours were subjected to three rounds of transfection with 50 nM of the different FITC-conjugated siRNAs for three hours at one-day intervals. (A) 80 hours after TPA treatment, transfection efficiency was determined by flow cytometry to detect FITC-positive cells. The histograms indicate green fluorescence (FL1) on the horizontal logarithmic axis. The controls are mock-transfected cells. (B) 40 µg of total protein extract from siRNA-treated cells was used for a western blot analysis of B-Myb protein levels. The upper panel shows the signal obtained after probing with anti-B-Myb, whereas the lower panel is an image of the Ponceau Red staining of the same region of the filter after transfer. (C) RT-PCR analysis of gene expression following siRNA knockdown of B-Myb. cDNA was prepared from 1 µg RNA extracted from cells treated as described in A. Semi-quantitative RT-PCR was performed for the indicated genes, sampling reactions at 22, 25 and 28 cycles, using HPRT to normalise the cDNA input.

 

Figure 5
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  		Fig. 5. Downregulation of B-Myb by RNA interference leads to a defect in S phase and the nuclear localisation of DNA replication. (A) Flow cytometric analysis of siRNA-transfected HEL cells cultured in the presence of TPA for 64 hours followed by 16 hours in the presence of TPA and BrdU. Incorporation of BrdU was detected by immunofluorescence using a FITC-conjugated mouse anti-BrdU monoclonal antibody and FITC-conjugated mouse IgG1 isotype control (FL1, vertical axis), and total DNA content was monitored by propidium iodide staining (FL2, horizontal axis). The histograms on the right show green fluorescence (FL1, horizontal axis) of 4C cells stained with a FITC-conjugated mouse anti-BrdU monoclonal antibody. The black-shaded histograms indicate the extent of anti-BrdU staining, whereas the grey lines show the staining obtained with the isotype control. (B) Immunofluorescence analysis of siRNA-transfected HEL cells grown in the presence of TPA for 64 hours and then pulse-labelled with BrdU for 45 minutes. Incorporation of BrdU was detected by indirect immunofluorescence using a mouse anti-BrdU antibody and PE-conjugated goat anti-mouse Ig secondary antibody (red). Nuclei were stained with DAPI (blue). Upper panels: control siRNA. Middle panels: B-myb siRNA1. Lower panels: B-myb siRNA2. (C) Histogram representing the pattern of incorporation of BrdU in 100 cells treated with each of the siRNAs. NM, no match.

 

Figure 6
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  		Fig. 6. Downregulation of B-Myb by RNA interference leads to a defect in chromosome condensation. HEL cells treated with 10–8 M TPA for 16 hours were subjected to three rounds of transfection with 50 nM of the different siRNAs for 3 hours at intervals of one day. At 2 hours after the final treatment, chromosome spreads were prepared in the presence of hypotonic buffer and stained with DAPI. (A) Control siRNA, (B) siRNA 1, (C) siRNA 2. The upper and lower panels show diploid and polyploid cells, respectively. The arrows indicate fragmented chromosomes.

 

Figure 7
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  		Fig. 7. Downregulation of B-Myb by RNA interference leads to chromosome instability. HEL cells treated with 10–8 M TPA for 16 hours were subjected to three rounds of transfection with 50 nM of the different siRNAs for 3 hours at intervals of one day. At 2 hours after the final treatment, chromosome spreads were prepared and stained using anti-CENPa (orange) and DAPI (green). Representative chromosome spreads are shown for cells treated with (A) control siRNA and (B) siRNA1 targeted against B-myb. The arrows in B indicate examples of aberrant chromosomes. (C) Expanded images of the types of chromosome aberration resulting from B-myb knockdown, including single chromatids (i,ii), chromosomes joined end-to-end (iii,iv), loss of chromosome segments (v,vi), and greater than two centromeres (vii,viii).

 

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© The Company of Biologists Ltd 2006