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Article Figures & Data

Figures

  • Fig. 1.

    RNA- and DNA-FISH analysis of polyploid megakaryocytes.

    Cells were cultured for 6 days and hybridized to different specific genomic probes. (A) Simultaneous RNA- and DNA-FISH analysis of polyploid megakaryocytes using FOG-1 and Fli-1 probes. RNA-FISH was performed using digoxigenin-labeled FOG-1 or Fli-1 probes revealed by an anti–digoxigenin FITC antibody (green) as described in “Materials and methods.” After image analysis, the cover slides were removed, the cells were washed in 4× SSC/0.1%Tween-20, and incubated in the presence of 0.1 mg/mL RNAse A for 30 minutes at 37°C. DNA-FISH was then performed after denaturation of cells in 70% formamide/2× SSC (3 minutes at 75°C) using the same digoxigenin-labeled FOG-1 or Fli-1 probes revealed by anti–digoxigenin TRITC antibody (red). Nuclear DNA was counterstained with DAPI (blue). The ploidy level determined from the number of red spots is indicated as 2N or 8N. Merging of the pictures obtained by RNA-FISH and DNA-FISH (merge) produced yellow spots, demonstrating the accumulation of nuclear FOG-1 or Fli-1 RNAs at the location of the corresponding gene. Note that most of the RNA and DNA spots superimpose in the case of the FOG-1, whereas the majority of RNA and DNA spots for Fli-1 were disposed side by side. (B) Effect of RNAse A or actinomycin D treatment on the signals obtained in RNA-FISH experiments. RNA-FISH experiments were performed with probes corresponding to the indicated genes. (i) The slides were treated (lower panel) or not (upper panel) with 0.1 mg/mL RNAse A for 30 minutes before hybridization. Absence of signals after RNAse treatment confirmed that the signals observed after hybridization without RNAse treatment correspond to RNA hybridization. (ii) The cells were incubated for 90 minutes in the absence (upper panels) or in the presence (lower panels) of 5 μg/mL actinomycin D (ActD) before being processed for RNA-FISH analysis. (C) Hsp70 RNAs detection. Megakaryocytes were heat-shock–treated by immersion for 30 minutes in a waterbath at 42°C or not treated (37°C). The number of transcription sites detected is indicated. Original magnifications × 60.

  • Fig. 2.

    Analysis of

    GPIIb or Fli-1 gene nuclear RNA accumulation and of ploidy level in polyploid megakaryocytes using confocal microscopy. Megakaryocytes were cultured for 8 days and double RNA- and DNA-FISH experiments were performed as in Figure 1A using a GPIIb or a Fli-1 probe (green) and a centromeric probe (red) staining chromosome 12 (Oncor, Gaithersburg, MD). Nuclear DNA was counterstained with DAPI (blue). The ploidy level determined from the number of red spots is indicated as 8N, 16N, or 32N. (A) Representative cells for each ploidy class. Original magnification × 63. (B) Spots corresponding to GPIIb or Fli-1 transcription sites were counted in 15 cells for each ploidy class using a confocal microscope. There were 3 laser excitation wavelengths used: 360 nm, 488 nm, and 543 nm, for DAPI, FITC, and TRITC, respectively. Serial optical sections of 2.5 μm (images collected at 0.25-μm intervals) in the z-axis of the cell were collected sequentially for each marker and overlaid to obtain a 2-dimensional reconstruction. Note that in megakaryocytes with a high ploidy level the spots were counted separately in serial optical sections through the cell. After superposition, 2 or 3 spots could yield a single strong signal. This approach allows a precise counting of the spots.

  • Fig. 3.

    Analysis of the accumulation of

    GATA-1 gene nuclear RNA and XIST RNA and of the ploidy level in polyploid megakaryocytes from male and female individuals.The experiments were performed and the results are presented as in Figure 1A except that a GATA-1 or XIST probe and a centromeric probe for chromosome X (Oncor) were used. M indicates male; F, female. 2N, 8N indicate the ploidy level. Original magnification × 60.