PCR primers for genotyping of ES cells and mice mutants.

<p>PCR primers for genotyping of ES cells and mice mutants.</p

Overall body and bone parameters in NM1 knock-out and wild type mice.

<p>Overall body and bone parameters in NM1 knock-out and wild type mice.</p

Preparation of NM1 knock-out cassette.

<p>(<b>A</b>) WT genomic locus of <i>Myo1c</i> gene. Short homology arm (SA), floxed part (FP), and long homology arm (LA) of appropriate length (0.9; 0.3; 1.7 kb respectively), were cloned to pEasyFlox vector carrying neomycin (NeoR) and thymidine kinase (ThK) selection genes (<b>B</b>). Black lines represent genomic sequences; red line represents sequences derived from pEasyFlox vector. (<b>B</b>). (<b>C</b>) Genomic loci of <i>Myo1c</i> gene with excision of exon -1; P1 – P6 represent different primers needed for genotyping of ES cells and knock-out mice, yellow triangles represent loxP recombination sites. (<b>D</b>) Genotyping of NM1 knock-out mice by PCR. P5 and P6 primers were used to distinguish between wild type (+/+), heterozygous (+/−) and knock-out (−/−) animals. (<b>E</b>) Western blot analysis of NM1 levels in NM1 wild type (+/+) and knock-out (−/−) mice. Fifteen micrograms of protein per lane was loaded, and probed for NM1. Equal loading was monitored by Coomassie Brilliant Blue staining of the band corresponding to actin.</p

Myo1C is able to function in Pol I and Pol II transcription without changes in its expression level.

<p>(<b>A</b>) The level of nascent rRNA was decreased to 80% of WT levels in NM1/Myo1c knock-down cells (U2OS+C8). An overexpression of mouse NM1 (U2OS+C8+NM1) or Myo1c (U2OS+C8+M1c) resistant to shRNA causes restoration of Pol I transcription to almost endogenous levels. As a negative control were used U2OS cells with transduced empty pLKO1.1 vector (U2OS+NC). (<b>B</b>) Both NM1-Flag and Myo1c-Flag interact with Pol II. Extracts from cells overexpressing NM1-Flag and Myo1c-Flag were co-immunoprecipitated with Flag antibody and control IgG. Immunoprecipitates were analyzed by western blotting with antibodies against Flag, Pol II CTD subunit and Myo1c (tail domain recognizing both NM1 and Myo1c). (<b>C</b>) NM1 knock-out does not cause compensatory changes in expression of Myo1c. Expression of Myo1c was measured by RT-qPCR and compared relative to GAPDH expression. Data are presented as mean +/− SD. *** p<0.001.</p

Ratio between NM1 and Myo1c is nearby equal.

<p>(<b>A</b>) HeLa cells were fractionated into cytosolic and nuclear fractions. NM1 and Myo1c amounts were quantified using double fluorescent labeling of western blot membranes after normalization to NM1-GFP band. (<b>B</b>) Total amounts of NM1+Myo1c were compared in mouse skin fibroblasts derived from NM1 knock-out and NM1 wild type mouse. Beta actin signal was used as loading normalizer. (<b>C</b>) Total amounts of NM1+Myo1c were compared in lungs and stomach from NM1 knock out and NM1 wild type mouse. GAPDH signal was used as loading normalizer. (<b>D</b>) The graph shows the amount of NM1 and Myo1c after densitometric quantification of bands from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061406#pone-0061406-g004" target="_blank">figures 4B and 4C</a> showing the ratio between NM1 and Myo1c as compared to actin/GAPDH expression.</p

NM1 knock-out has no effect on cell proliferation and Pol I transcription.

<p>(<b>A</b>) Proliferation of NM1 KO cells (NM1 −/−) is not altered in comparison to WT cells (NM1 +/+). 1×10⁵ cells were seeded on plates (20% confluence; day 0) and let to grow for six days when number of cells was counted again (day 6). (<b>B</b>) Pol I transcription rate in NM1 KO (NM1 −/−) and WT (NM1 +/+) cells is equal. RNA from exponentially growing cells was isolated and expression of 45S pre-rRNA was measured by RT-qPCR. Expression of 45S pre-rRNA is compared relatively to GAPDH expression. Data are presented as mean +/− SD.</p

Red blood cells related phenotype in NM1 knock-out mice.

<p>Red blood cells related phenotype in NM1 knock-out mice.</p