MS data for Huang et al. NCB paper

<p>To identify cofactors of IGF2BPs, we ectopically expressed FLAG-IGF2BP2 in HEK293T cells, pulled down the IGF2BP2 complexes using FLAG antibody and conducted mass spectrometry analysis. Immunoprecipitaion with IgG control and the subsequent mass spectrometry analysis were perform in parallel as a negative control. Proteins identified with 4 or more unique peptides each at 99% confidence are listed in the "Processed data.xlsx" file.</p><p></p

Influenza virus induced cell-cell fusion. MDCK cells were infected with the viruses at MOI of 0.1 or 0.001 in the presence of 1 µg/ml TPCK-trypsin.

<p>After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated for the indicated times at 37°C in the maintenance media. At the indicated time, the cells were processed for indirect immunofluorescence assay, and the infected cells were detected with polyclonal antisera to whole viruses. (A) MOI at 0.1, 3 h p.i. (B) MOI at 0.1, 6 h p.i. (C) MOI at 0.001, 12 h p.i.</p

vtRNA1-1 promotes the tumor formation and enhances the chemoresistance of DOX in vitro.

<p>(A) After transfection of vtRNA1-1, RBD or vtRNA1-1/RBD, sensitivity to DOX was measured in MCF-7 cells. (B) the expressing levels of GAGE6 and PSF after the DOX treatment were detected in MCF-7 transfected with vtRNA1-1, RBD or vtRNA1-1/RBD and the relative folds were qualified (C). (D) ChIP was performed to detect the binding of PSF to GAGE6 promoter in MCF-7 transfected with vtRNA1-1, RBD or vtRNA1-1/RBD after DOX exposure. CCK-8 assay (E) or PI staining (F) was performed to detect cell viability in MCF-7 transfected with vtRNA1-1 after DOX exposure. *P<0.05.</p

Impact of different NA on initiation of influenza virus infection.

<p>MDCK cells were infected at an MOI of 0.001 in the presence of 1 µg/ml TPCK-trypsin. After adsorption for 1 h at 37°C, the inocula were removed and the cultures were washed 3 times. The cells were incubated at 37°C for 6 h. At the indicated time, the cells were processed for immunofluorescence, and the infected cells were detected with polyclonal antisera to whole viruses. (A) Fluorescence images of the infected cells at 6 h p.i. Fluorescent photomicrographs showing the intracellular expression of virus protein in cell culture. The FITC-fluorescence signal was expressed as the infected cells. (B) Volocity Demo software analysis of the ratios of infected cells according to the Fig. 5A. *, statistical significance (<i>p</i><0.05) (C) Flow-cytometric analysis of virus-infected cells at 6 h p.i. MDCK cells (2×10⁶) in suspension were incubated with PBS or anti-PR8 antibodies on ice. Then the FITC-conjugated IgG secondary antibodies were added. After washing, the cells were fixed and the number of infected cells was determined by flow cytometric analysis. *, statistical significance (<i>p</i><0.05).</p

VRNA binds to PSF on its RNA binding domain (RBD) and releases GAGE6 promoter from PSF in vitro.

<p>(A) Northern blot was performed to detect the existence of vtRNA1-1, 1–2, 1–3 or 2–1. (B) qRT-PCR analysis was performed to quantify the relative amount of vtRNA1-1, 1–2, 1–3 or 2–1 normalized to 5.8S rRNA. (C) EMSA analysis was performed the binding of vtRNA1-1, 1–2, 1–3 or 2–1 to purified PSF in vitro. (D) The specific binding of vtRNA1-1 to RBD of PSF was detected. (E) the competitive binding of vtRNA1-1 to PSF against GAGE6 promoter fragment was analyzed.</p

The effects of vtRNA1-1 in cell proliferation and colony formation in a PSF-dependent way.

<p>Cell curve assays were performed to identify the association of vRNA with PSF (A, left panel), RBD or DBD (B, right panel) on affecting the cell proliferation in MCF-7. (B) EdU staining was performed to further confirm the effect of vRNA on cell proliferation. (C) Soft agar assay was performed to present vRNA’s effect on colony formation in MCF-7. *P<0.05.</p

vtRNA1-1 expression tightly regulates the expressing level of GAGE6 in MCF-7 and MCF-7/MR.

<p>(A) RNA-IP assay confirmed the specific binding of PSF to vtRNA1-1 through it RBD in vivo. RT-qPCR (B) and semi-quantitative western blot (C) presents the expressing changes in the mRNA and protein level of GAGE6 in MCF-7 and MCF-7/MR. Chromatin immunoprecipitation assays present that the binding capacity of PSF to GAGE6’s promoter region affected by vtRNA1-1 in MCF-7 (D) or MCF-7/MR (E). RT-qPCR (F) and Semiquantitative western blot (G) present the mRNA and protein levels affected by the ectopic expression of vtRNA1-1 in MCF-7 or MCF-7/MR. *P<0.05.</p

Virus elution in <i>vitro</i>.

<p>50 µl two-fold dilutions of virus containing the HA titers of 1∶128 was incubated with 50 µl 0.5% chicken erythrocytes in microtiter plates at 4°C for 1 h. Then microtiter plates were incubated at 37°C, and the reduction of HA titers was measured periodically for 8 h.</p

Alignment of the deduced amino acid sequences of the NA genes from the influenza virus strains A/Chicken/Jiangsu/7/2002(H9N2), A/California/04/2009 (H1N1), A/chicken/Henan/12/2004 (H5N1) and PR8 (H1N1).

<p>Alignment of the deduced amino acid sequences of the NA genes from the influenza virus strains A/Chicken/Jiangsu/7/2002(H9N2), A/California/04/2009 (H1N1), A/chicken/Henan/12/2004 (H5N1) and PR8 (H1N1).</p

vtRNA1-1 induced mitoxantrone independent with the existence of MVP.

<p>(A) The efficiency of MVP or vtRNA1-1 knockdown was detected by semiquantitative western blot or qRT-PCR. (B) DOX sensitivity to MCF-7 cells with or without vtRNA1-1 knockdown was detected. (C) Immunofluorescence imaging was performed to detect the sub-cellular localization of DOX with or without vtRNA1-1 knockdown.</p