ythdf3 targeting sirna  (Santa Cruz Biotechnology)

Journal: Nature microbiology

Article Title: N 6 -methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I

doi: 10.1038/s41564-019-0653-9

Figure Lengend Snippet: (a) Immunostaining spots formed by rhMPVs. (b) Quantification of m 6 A level . Total m 6 A level of each virion RNA was quantified by m 6 A RNA Methylation Assay Kit. (c) m 6 A-deficient rhMPV RNA has reduced binding efficiency to reader proteins . Cell lysate containing HA-tagged YTHDF1 or YTHDF2 was incubated with virion RNA and anti-HA Magnetic beads. The amount of virion RNA captured by the YTHDF1 or YTHDF2 was quantified by real-time RT-PCR. Percent of bound RNA of hMPV mutants relative to rhMPV was calculated. IFN-β secretion in A549 cells infected by hMPV at MOI of 4.0 (d, h) or an MOI of 1.0 (e). A549 cells were infected with each rhMPV at an MOI of 4.0, and IFN-β in cell supernatants were measured by ELISA. Dynamics of IFN-β secretion in THP-1 cells infected by hMPV at an MOI of 4.0 (f-g). (i) IFN - β response in A549 cells transfected with total RNA. Total RNA was extracted from hMPV-infected A549 cells, and the antigenome was quantified by real-time RT-PCR. A549 cells were transfected with 10 8 antigenome RNA copies of total RNA with or without treatment CIP. IFN-β was measured by ELISA. (j) IFN - β response in A549 cells transfected with viral G mRNA. A549 cells were transfected with 10 9 RNA copies of G mRNA either with or without CIP treatment. (k) IFN - β response in A549 cells transfected with virion RNA. A549 cells were transfected with 2×10 7 antigenome copies of virion RNA either with or without CIP treatment. (l and m) Comparison of IFN response of virion RNA of rhMPV-G1-14, G1-2, G8-9, and rhMPV. A549 cells were transfected with 10 7 (l) or 10 6 (m) RNA copies of virion RNA. (n and o) Natural m 6 A-deficient virion RNA induces IFN response. A549 cells were transfected with 10 7 (n) or 10 6 (o) RNA copies of virion RNA of rhMPV-G1-14, G(-)1-6, ALKBH5, and rhMPV. Immunospots ( a ) shown are the representatives of n = 3 biologically independent experiments. Data shown are means of n = 3 ( c-o ) or n = 4 ( b ) biologically independent experiments ± standard deviation. Statistical significance was determined by two-sided student’s t -test. Exact P values are included in Data Source. * P <0.05; ** P <0.01; *** P <0.001; **** P <0.0001, NS, no significant.

Article Snippet: siRNAs against METTL3, METTL14, FTO, ALKBH5, YTHDF1, YTHDF2, YTHDF3 or non-targeting AllStars negative control siRNA were purchased from Qiagen (Valencia, CA, sequences listed in ).

Techniques: Immunostaining, Methylation, Binding Assay, Incubation, Magnetic Beads, Quantitative RT-PCR, Infection, Enzyme-linked Immunosorbent Assay, Transfection, Comparison, Standard Deviation

Journal: Nature Communications

Article Title: Viral N 6 -methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

doi: 10.1038/s41467-019-12504-y

Figure Lengend Snippet: Effects of m 6 A eraser proteins on RSV gene expression. a Overexpression of m 6 A eraser proteins diminishes RSV gene expression. HeLa cells were transfected with plasmids encoding ALKBH5 and/or FTO, followed by rgRSV infection at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for Western blot analysis. b Overexpression of m 6 A eraser proteins reduces GFP expression. The GFP expression was monitored by fluorescence microscopy. Representative micrographs with 10 × magnification (scale bar of 100 μm) at 18 h post-infection were shown. c Quantification of GFP-positive cells by flow cytometry at 18 h post-infection. The P value (Student’s t-test) for ALKBH5, FTO, and ALKBH5&FTO is **** P = 1.91 × 10 −6 , **** P = 1.34 × 10 −5 , and **** P = 3.61 × 10 −6 , respectively. d Knockdown of m 6 A eraser proteins enhances RSV gene expression. HeLa cells were transfected with siRNA targeting ALKBH5 and/or FTO, followed by rgRSV infection at an MOI of 0.5. At 18 h post-infection, cell lysates were harvested for Western blot analysis. e Knockdown of m 6 A eraser proteins enhances GFP expression. Micrographs with 10 × magnification (scale bar of 100 μm) are shown. f Quantification of GFP-positive cells by flow cytometry. Fold of GFP signal compared to the control is shown. Western blots and GFP images shown are representatives of three independent experiments. Flow cytometry data are expressed as mean ± standard deviation. The P value (Student’s t -test) for ALKBH5, FTO, and ALKBH5&FTO is **** P = 2.06 × 10 −4 , **** P = 3.38 × 10 −5 , and **** P = 6.50 × 10 −6 , respectively. g Overexpression of m 6 A eraser protein reduces m 6 A level in viral RNA. A549 cells were transfected with plasmid encoding ALKBH5 or pCAGGS, followed by rgRSV infection. RSV particles were purified from supernatants. Virion RNA was subjected to m 6 A-IP. The P value (Student’s t -test) is **** P = 5.81 × 10 −9 . h Fold reduction in m 6 A content in viral RNA compared to controls. The P value (Student’s t -test) is **** P = 1.17 × 10 −11 . i Knockdown of m 6 A eraser protein increases m 6 A level in viral RNA. A549 cells were transfected with siRNA targeting ALKBH5 or control siRNA, followed by rgRSV infection. RSV particles were purified. Virion RNA was subjected to m 6 A-IP. The P value (Student’s t -test) is **** P = 5.09 × 10 −9 . j Fold increase in m 6 A content in viral RNA compared to controls. The P value (Student’s t -test) is **** P = 2.57 × 10 −6 . Results are from three or four independent experiments

Article Snippet: siRNAs against METTL3, METTL14, FTO, ALKBH5, YTHDF1, YTHDF2, YTHDF3, or non-targeting AllStars negative control siRNA were purchased from Qiagen (Valencia, CA, sequences listed in Supplementary Table ).

Techniques: Gene Expression, Over Expression, Transfection, Infection, Western Blot, Expressing, Fluorescence, Microscopy, Flow Cytometry, Knockdown, Control, Standard Deviation, Plasmid Preparation, Purification

Journal: Nature Communications

Article Title: Viral N 6 -methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

doi: 10.1038/s41467-019-12504-y

Figure Lengend Snippet: RSV infection does not alter the m 6 A reader, writer, or eraser protein distribution in cells. HeLa cells were infected by rgRSV at an MOI of 10.0. At 24 h post-infection, mock- or rgRSV-infected cells were stained with anti-reader, writer, or eraser protein antibody (green) and anti-RSV N protein antibody (red), and were analyzed by confocal microscope. Nuclei were labeled with DAPI (blue). Micrographs with ×60 magnification (scale bar of 20 μm) are shown. a m 6 A reader protein YTHDF2; b m 6 A writer protein METTL3; and c m 6 A eraser protein FTO. d Detection of m 6 A reader, writer, and eraser proteins by Western blot. Nuclear and cytoplasmic fractions were separated from mock- or rgRSV-infected HeLa cells, and were subjected to western blot. Nuclear and cytoplasmic markers were indicated by Lamin A and α-Tubulin, respectively. Representative results from three independent experiments are shown

Article Snippet: siRNAs against METTL3, METTL14, FTO, ALKBH5, YTHDF1, YTHDF2, YTHDF3, or non-targeting AllStars negative control siRNA were purchased from Qiagen (Valencia, CA, sequences listed in Supplementary Table ).

Techniques: Infection, Staining, Microscopy, Labeling, Western Blot