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Data supplements

  • Supplemental materials for: Bolli et al

    Files in this Data Supplement:

    • Figure S1 (JPG, 428 KB) -
      (A, B) Confocal microscopy of DsRed-Npm1a� and DsRed-Npm1b�injected embryos showing nucleolar localization in cells from whole zebrafish embryo preparations at 24 hpf. 10 pg of each mRNA were used for injections. Npm1a (A) and Npm1b (B) are shown in red, while nuclei are counterstained in blue with DAPI. Images of cells were taken in the dorsal trunk region with a Zeiss LSM 510 META 2-Photon confocal microscope (Carl Zeiss), using a Zeiss 63×/1.4 NA Apochromat Oil lens (Carl Zeiss). Images were acquired with the Zeiss LSM 510 software (Carl Zeiss). The DsRed expression patterns shown were representative of cells throughout the embryo. (C, D) lateral views of 2 dpf embryos, anterior to the left dorsal upwards. WISH assays for mpx in control injected (C, 16ng) and npm1a+npm1b splice MO injected (D, npm1a E2-I2(2), 8ng +, npm1a E3-I3, 4ng + npm1b E2-I2, 4ng) embryos. Reduced numbers of mpx-expressing cells are observed in D. (E) shows quantification of the reduction in 15 embryos per condition. (p<0.0001; unpaired Student�s t-Test. F) RT-PCR genotyping using full length primers for npm1a (left) and npm1b (right) coding sequences. Sequencing of aberrant splice products (delineated by the white boxed lane) demonstrated truncating splice products at the sites indicated by arrows. SD=splice donor, E=exon, I=intron.





    • Figure S2 (JPG, 200 KB) -
      (A) NPMc+ dislocates NPM1 from nucleoli to the nucleoplasm and cytoplasm. Epifluorescent images of 293T cells co-transfected with pDsRed-monomer-C1-NPM1 (in red, left panel) and pEGFP-C1-NPM1c+ (in green, middle panel). Co-localization areas between NPMc+ and NPM1 are shown in yellow in the merged images (right panel). Nuclei are counterstained in blue with DAPI. Images were acquired with a Zeiss Axio imager Z1 microscope using a Zeiss 63×/1.4 NA Apochromat Oil lens (Carl Zeiss) and Openlab software (Perkin Elmer). (B, C) NPM1 and NPMc+ co-precipitate Npm1a and Npm1b. (B) Western Blot showing anti-FLAG immunoprecipitation of protein lysates from 293T cells transfected with empty FLAG-vector and either EGFP-Npm1a or Npm1b. Left two lanes: input. Right two lanes: anti-FLAG co-IP showing no aspecific binding of Npm1a or Npm1b to the FLAG tag. No FLAG band is shown due to the extremely small size of the tag (~4kD). (C) WB showing anti-FLAG immunoprecipitation of protein lysates from 293T cells transfected with FLAG-NPM1 or FLAG-NPMc+ and either EGFP-Npm1a or Npm1b. Left four lanes: input. Right four lanes: anti-FLAG co-IP showing that both NPM1 and NPMc+ can bind either npm1a or npm1b.





    • Figure S3 (JPG, 282 KB) -
      (A�F) FACS analysis of annexin V-PE binding in single cell suspensions from Tg(pu.1:EGFP) 24 hpf embryos, uninjected (A, D) or injected with NPM1 10 pg (B, E) or NPMc+ 50 pg (C, F). Total cells are shown in A�C, and cells expressing EGFP under the control of the pu.1 promoter are shown in D�F. NPMc+ slightly increases the number of total cells undergoing apoptosis (C), but not significantly in pu.1:EGFP positive cells (F). (G�I) Propidium Iodide cell cycle analysis in sorted cells expressing EGFP under the control of the pu.1 promoter from Tg(pu.1:EGFP) 24 hpf embryos, uninjected (G) or injected with NPM1 10 pg (H) or NPMc+ 50 pg (I). No alterations of the cell cycle profiles were observed upon injection of the NPM1 or NPMc+ RNA.





    • Figure S4 (JPG, 792 KB) -
      Ai�iii, Ei�iii, Ii�iii, M) WISH assays (i) showing lateral views (anterior to the left, dorsal upwards) of embryos stained for c-myb at 32 hpf, uninjected (Ai), or injected with NPM1 10 pg (Ei) or NPMc+ 50 pg (Ii) mRNA. Cross sections of c-myb�stained embryos (ii) show c-myb cells are expressed correctly at the midline below the aorta and above the cardinal vein (magnified in -iii). Scale bar in Iii= 25 um. M) c-myb positive cell number quantification shows a statistically significant increase upon NPMc+ expression (20 embryos per condition). (B, F, J, N) FACS analysis of Tg(c-myb:EGFP) embryos, uninjected (B), or injected with NPM1 10 pg (F) or NPMc+ 50 pg (J) mRNA shows that EGFP expression (Y-axis) is increased in NPMc+ injected embryos (J). N) Quantification of the percentage of EGFP+ cells in three independent FACS experiments, each including 30 embryos per condition, normalized to uninjected controls. FACS analysis has been conducted in dissected trunks of c-myb:EGFP+ embryos only including FSCHIGH cells as described in24gata1:EGFP);(lmo2:DsRed) transgenic zebrafish embryos, uninjected (C), or injected with NPM1 10 pg (G) or NPMc+ 50 pg (K) mRNA shows that the percentage of gata1+/lmo2BRIGHT is increased in NPMc+ injected embryos (K). O) Quantification of the percentage of double EGFP+;DsRed cells in three FACS replicates, each including 30 embryos per condition, normalized to uninjected controls. FACS analysis has been conducted as described in.23 (D, H, L, P). Lateral view (anterior to the left, dorsal upwards) of the trunk region of 32hpf p53m∕m embryos, uninjected (D) or either injected with NPM1 10 pg (H) or NPMc+ 50 pg (L) mRNA and stained for c-myb expression. c-myb expressing cells are markedly expanded upon NPMc+ expression (L), and were quantified in (P) by counting cells in 15 embryos per condition. Error bars represent standard error of the mean. Asterisks indicate statistically significant differences between the NPMc+ injected and both NPM1 and uninjected embryos (*=p<0.05; Student�s t-Test). In histograms blue=uninjected control embryos, green=NPM1-injected embryos, red=NPMc+ injected embryos.





    • Figure S5 (JPG, 361 KB) -
      (A�I) Lateral view (anterior to the left, dorsal upwards) of the trunk region of 28 hpf (A, D, G) and 32hpf (B, C, E, F, H, I) embryos. Embryos are uninjected (A�C) or either injected with NPM1 10 pg (D�F) or NPMc+ 50 pg (G�I) mRNA and stained for runx1 (A�B, D�E, G�H) or gata-1 (C, F, I). No difference in runx1 and gata1 expression were observed between the different conditions.