Grisendi et al.
Supplementary Figure 1 Targeted disruption of the Npm gene by homologous recombination. a, Structure of the mouse wt Npm locus (top), the targeting vector (middle) and the targeted allele (bottom). Exons 2 to 7 are replaced by a GFP cDNA sequence in frame with exon 2. The NEO resistance is under the control of the pGK promoter. NEO is excised through Cre-mediated recombination between two loxP sites (triangles). Angiotensin-converting enzyme gene promoter (pACE) drives the expression of Cre recombinase in spermatocytes, leading to deletion of the NEO and Cre sequences in the mutant mice germ line. Position of the 5’ and 3’ probes used to confirm the homologous recombination is shown. Primers pA, pB and pC have been used for genotyping. E, EcoRI; S, SacI; P, PmeI; N, NcoI; Pa, PacI and A, AscI. b, Southern blot analysis on EcoRI and SacI digested genomic DNA with 5’ and 3’ external probes demonstrates homologous recombination in ES cells. c, Genotyping of the mutant mice by multiplex PCR on tail genomic DNA using the indicated primers demonstrates germ line transmission and deletion of the NEO cassette. d, Western blot analysis of Npm expression in MEFs of the indicated genotypes. e, H&E staining of sagittal sections from wt and Npm-/- E10.5 embryos (upper panels), and E10 embryos GFP fluorescence in cryo-sections (lower panels). f, Npm and GFP expression in E8.5 and E9.5 wt and Npm-/- embryos. g, Npm-/- embryos have a smaller but normally organized placenta. Comparable sections of E8.5 wt and Npm-/- embryos show normally developed allantois (arrowheads) in the process of reaching and fusing to the corion (arrows). h, At the interface between maternal (arrowheads) and fetal blood (arrows), embryonic erythroblasts are reduced in number in the Npm-/- embryos. i, CD34 immunostaining of E9.5 wt and Npm-/- embryo sections, Scale bars: 500 mm in e, 50 mm in i.
Supplementary Figure 2 Generation of Npm hypomorphic mice. a, Schematic representation of the targeted hypomorphic Npm allele. A removable pGK-NEO cassette has been inserted in the intronic sequence between exons 6 and 7 of the gene (grey ovals indicate Frt sites). Deletion of the first half of the gene could also be obtained through Cre-mediated recombination (black triangles indicate loxP sites). 5’ and 3’ probes used to confirm the homologous recombination and to genotype the mutant mice are shown. E, EcoRI; P, PvuII, H, HindIII; D, DraI; Pa, PacI. Parentheses indicate loss of the original restriction site. The lower panels show southern blot analysis on PvuII and HindIII digested genomic DNA with 5’ and 3’ external probes demonstrating homologous recombination in ES cells. b, The NEO cassette causes transcriptional interference. Npm expression in the hypomorphic series by northern blot (left panel) and western blot (middle panel) analysis of Npm+/+, Npm+/-, Npmhy/hy and Npm-/- MEFs. c, Wt and Npmhy/hy E13.5 embryos: similarly to Npm-/- mutants, Npmhy/hy embryos are smaller in size, pale and generally well formed except for the anterior brain and the eye. d, Sagittal sections of E13.5 wt and Npmhy/hy embryos show up-regulation of p53 expression in the hypomorphic mutant (arrows). Scale bar: 250 mm. Right panels show 10X magnifications of the neural tissues. e, Ribosome profile of Npm deficient MEFs. Reduction in Npm expression levels affects the relative amounts of the ribosomal subunit 80S in Npm+/- cells, while in Npmhy/hy cells the relative amount of all the subunits (40S, 60S and 80S) is decreased compared to a wt controls.
Supplementary Figure 3 Analysis of apoptosis and proliferation in E9.5 wt and Npm-/- embryos. a, Deletion of the Npm gene induces an apoptotic response with increase of activated caspase-3 expression, while Ki-67 proliferation marker is comparable in wt and mutant embryos. Scale bars: 500 mm. b, Percentages of activated caspase-3 and Ki-67 expressing cells in the neuroepithelium of wt and Npm-/- embryos.
Supplementary Figure 4 Npm is haploinsufficient for maintenance of genomic stability and tumour susceptibility. a, Chromosome FISH analysis of Npm deficient MEFs. Number of wt, Npm+/- and Npmhy/hy cells with the indicated number of FISH signals per cell are shown. b, Growth curves of wt and Npm+/- P4 MEFs in a wt and p53-/- background. Values are the average results from 3 different MEF preparations per genotype. c, Soft agar colony forming assay on wt and Npm+/- MEFs at the indicated passage number, concomitantly infected with RasV12 and E1A or the control empty vectors. d, CGH Array analysis of lymphomas from Npm+/+ Em-Myc and Npm+/- Em-Myc mice. Chromosomal abnormalities detected in lymphomas from Npm+/+ Em-Myc (n=4) and Npm+/- Em-Myc mice (n=4) are shown. In some cases, different lymphomas from the same mouse were analyzed, as indicated. Chromosome gains and losses are listed as numerical abnormalities. The presence of structural abnormalities, such as deletion and duplication of chromosome regions, is indicated for each mouse. Note that chromosome 11, where murine Npm maps to, was never found lost or rearranged. e, LOH analysis on tail (T) and tumour (Tu) DNA derived from Npm+/+ Em-Myc and Npm+/- Em-Myc mice. DNA was analyzed by southern blot hybridization with the 5’ probe utilized to identify the Npm null allele (see also sFig. 1). Two representative cases are shown. The Npm genotype is indicated at the bottom of the panels. f, Expression of the Npm protein is retained in tumors from both Npm+/+ Em-Myc and Npm+/- Em-Myc mice as indicated by the immunohistochemical analysis of Npm in lymphomas isolated from Em-Myc mice with the indicated Npm genotype.
Supplementary Figure 5 Npm+/- mice display myelodysplastic features. a, Dyserythropoiesis in Npm+/- mice: representative images of BM samples from Npm+/- mice, arrows point to cells with cytoplasmic blebs and nuclear fragmentation. b, Dysplastic megakaryocites: representative images of BM samples from one wt and one Npm+/- mouse show increased number of megakaryocytes in Npm+/- mice (see text for details); arrows point to cells with abnormally small hypolobated nuclei. c, Bone marrow FACS analysis summary table illustrating the number of mice analyzed and the average values obtained (see also Fig.4d and e). d, E/M relative ratio in wt and Npm+/- mice, evaluated as ratio between the number of erythroid and myeloid precursors in the BM. For each mouse, 5 independent counts of total BM precursors (n=100) were performed.
3