Figure 1.
Preparation of NM1 knock-out cassette.
(A) WT genomic locus of Myo1c 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). Black lines represent genomic sequences; red line represents sequences derived from pEasyFlox vector. (B). (C) Genomic loci of Myo1c 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. (D) Genotyping of NM1 knock-out mice by PCR. P5 and P6 primers were used to distinguish between wild type (+/+), heterozygous (+/−) and knock-out (−/−) animals. (E) 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.
Table 1.
PCR primers for genotyping of ES cells and mice mutants.
Table 2.
Lung function parameters tested in NM1 knock-out and wild type mice.
Figure 2.
Red blood cells related phenotype in NM1 knock-out mice.
Table 3.
Overall body and bone parameters in NM1 knock-out and wild type mice.
Figure 3.
NM1 knock-out has no effect on cell proliferation and Pol I transcription.
(A) 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) 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.
Figure 4.
Ratio between NM1 and Myo1c is nearby equal.
(A) 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) 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. (C) 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. (D) The graph shows the amount of NM1 and Myo1c after densitometric quantification of bands from figures 4B and 4C showing the ratio between NM1 and Myo1c as compared to actin/GAPDH expression.
Figure 5.
Myo1C is able to function in Pol I and Pol II transcription without changes in its expression level.
(A) 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) 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). (C) 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.