Although NPM1.2 expression in human is only detected in HeLa cells which stably express human papillomavirus (HPV) E6 and E7 proteins (Dalenc et al., 2002; Sautkina et al., 2008), the function is largely unclear (Herrera, Savkur, and Olson, 1995; Savkur and Olson, 1998). hexon, L2 and L3; major late transcript units. (B) Schematic representation of the physical map of Ad5 and the adenoviral mutants used in this study. TP; terminal protein, E1; early gene 1, IX; polypeptide IX, mRFP1; monomeric red fluorescent protein 1, EGFP; enhanced green fluorescent protein, E3; early region 3, E1b19k; the gene for early gene 1b (E1b) 19 kilodalton protein, dE1; the E1 deletion, dV; the pV deletion, and dE3; the E3 deletion. NIHMS391973-supplement-SFig01.tif (17M) GUID:?D0547C49-751F-4829-A7D1-E2B0C4A51C5D SFig02: Supplementary Fig. 2. Ad5-dV/TSB replication is usually greatly restricted in 293 cells 293 cells were infected with Ad5 or Ad5-dV/TSB at an MOI of 10 PFU/cell for various times. One-step growth curve analysis of adenoviruses was performed, and infectious titer was determined by plaque assay. NIHMS391973-supplement-SFig02.tif (2.1M) GUID:?F46B7A03-EC93-40B1-B191-E9EF1BAA0777 SFig03: Supplementary Fig. 3. Optimization of immunofluorescence staining in cells (A) After medium was removed from HPAEC cells, cells were washed with 30 mM HEPES-NaOH buffer (pH 7.4) and fixed with fixation solution. HPAEC cells were stained with anti-NPM1 or anti-p53 followed by a secondary antibody conjugated with NHS-Biotin fluorescent probe. (B) After medium was removed from HPAEC and MCF-7 cells, cells were directly fixed with fixation solution without washing. Subcellular localization of NPM1 and p53 was determined by immunofluorescent staining. Hoechst 33342 staining was performed for 10 min. NIHMS391973-supplement-SFig03.tif (12M) GUID:?AFF6E3DB-11F7-468A-A93F-1CB11BADADDD SFig04: Supplementary Fig. 4. Adenoviral virion-associated core protein V-EGFP (pV-EGFP) induces disruption of NPM1 nucleolar localization (A) Detection of the fluorescently labeled adenoviral particles in infected HPAEC cells. HPAEC cells were infected with Ad5-E3-V-EGFP at MOIs 10, 100 or 1,000 PFU/cell for 1 hour. The trafficking of virion-associated core pV-EGFP was monitored in infected cells at 1 h.p.i. Uninfected and infected HPAEC cells were stained with anti-NPM1 followed by a secondary antibody conjugated with fluorescent probe. (B) Localization of NPM1-EGFP in 293 cells stably expressing NPM1-EGFP. Hoechst 33342 staining was performed for 10 min, and the signals for NPM1-EGFP and DNA staining were detected by fluorescence microscopy. NIHMS391973-supplement-SFig04.tif (13M) GUID:?D6C10777-2FA1-42FD-8B1B-F059B9AC99CC SFig05: Supplementary Fig. 5. Expression level of NPM1 in cancer and primary cells, and viral DNA replication in cancer cells (A) Crude proteins were extracted from HUVEC, HPAEC, and lung carcinoma A549 cells, and 10 g of total proteins were separated on 10% (w/v) SDS-PAGE, and NPM1 expression level was validated by Western blot analysis. (B) DNA replication of Ad5-dV/TSB in cancer cells. A549 cells were infected with Ad5 or Ad5-dV/TSB at an MOI of 10 NHS-Biotin PFU/cell, and harvested at various times post-infection. Total DNA was extracted from infected cells, and viral DNA replication was analyzed by using 50 ng of total DNA and E4 primers. Plots represent as the copy number of the E4 gene per 50 ng of total DNA. NIHMS391973-supplement-SFig05.tif (4.2M) GUID:?B1DDE2C3-583F-4610-8C5A-94A386CB62B5 SFig06: Supplementary Fig. 6. Transient expression of pV induces NPM1 and p53 redistribution and enhanced adenoviral production (A) HPAEC was infected with pV-mRFP1-lentivirus at an MOI of 50 proviral DNA/cell for 60 hours. Subcellular localization of NPM1 NHS-Biotin and p53 was detected in HPAEC infected with mock NHS-Biotin (LV-empty) or pV-recombinant lentivirus (LV-V). (B) Western blot analysis of pV expression in pV-lentivirus-infected HPAEC cells. (C) HPAEC cells were infected with the pV-recombinant lentivirus at an MOI of 2, 10 or 50 proviral DNA/cell for 60 hours. The pV-transduced cells were infected with adenovirus at an MOI of 10 PFU/cell, and harvested at 1 and 48 h.p.i. Production level of infectious adenovirus was measured by plaque assay. Results were reported as mean SD. * (Matthews, 2001). Also, adenoviral contamination induces NPM1 nucleolar delocalization (Walton et al., 1989). NPM1 is an abundant nucleolar phosphoprotein (Grisendi et al., 2006), but is usually expressed at fairly modest levels in human normal cells (Nozawa et al., 1996). In contrast to LATS1 the expression of NPM1 in human normal NHS-Biotin cells, NPM1 is usually isolated as one of the most abundant nuclear matrix proteins in cancer cells (Mattern et al., 1996; Zink, Fischer, and Nickerson, 2004), and detected in the nucleoplasm as well as in the nucleoli of tumors (Subong et al., 1999) and overexpressed in various types of tumors (Grisendi et al., 2006). Thus, the localization and expression of NPM1 are altered in human cancers (Grisendi et al., 2006). On the other hand, NPM1.2 which lacks the 35 amino acids at the C-terminus of NPM1, encodes the RNA binding site, RNase domain name, and nucleolar localization signal of NPM1 (Herrera, Savkur, and Olson, 1995; Hingorani, Szebeni, and Olson, 2000; Wang, Umekawa, and Olson,.
