Journal: Nucleic Acids Research

Article Title: Nuclear AGO2 supports influenza A virus replication through type-I interferon regulation

doi: 10.1093/nar/gkaf268

Figure Lengend Snippet: Tetrameric p53 protects nuclear AGO2 from proteasomal degradation ( A ) Schematic diagram of human p53 protein and N -terminal truncated Flag-tagged p53 isoforms used in the study. ( B ) Representative Flag immunoblots from HEK293 cells transfected with Flag-WT-p53 or N -terminally Flag-tagged p53 mutants. GAPDH served as a loading control. n = 3 ( C ) Flag IP from HEK293 cells transfected with plasmids expressing Flag-WT-p53 or N -terminally Flag-tagged p53 mutant. HEK293 cells were transfected with the p53 plasmids for 1 day and then treated with 1 μg/mL Doxorubicin (Doxo) for 1 more day. Representative immunoblots of AGO2 and Flag are indicated. n = 3 ( D ) Representative AGO2 and p53 immunoblots from cytoplasmic (C) and nuclear (N) lysates in A549 cells treated with 0.5 μg/mL arsenic trioxide (ATO) for 24 h. GAPDH served as a cytoplasmic marker and HIST3H3 served as nuclear marker. n = 3 ( E ) p53 IP from A549 cells treated with 0.5 μg/mL Arsenite trioxide (ATO) for 24 h. Representative immunoblots of AGO2 and p53. n = 3 ( F ) Docking model of the PIWI domain of AGO2 with the N -terminal region of p53 was generated using the HDOCK server. The PIWI domain is depicted in green, and the N -terminal region of p53 is shown in pink (model 4) in ribbon representation. ( G ) AGO2 IP from cytoplasmic (C) and nuclear (N) lysates in A549 cells. Representative immunoblots of AGO2, p53 and TNRC6A. n = 3 ( H ) p53 IP from cytoplasmic (C) and nuclear (N) lysates in A549 cells. Representative immunoblots of AGO2, p53 and TNRC6A. n = 3 ( I ) TNRC6 IP from cytoplasmic (C) and nuclear (N) lysates in A549 cells treated with siRNAs for 24 h. siCntr: control scramble siRNAs; and siAGO2: siRNA specific for AGO2. Representative TNRC6, AGO2 and p53 immunoblots. GAPDH served as a cytoplasmic marker and HIST3H3 served as nuclear marker. ( J ) Representative AGO2 and p53 immunoblots from cytoplasmic (C) and nuclear (N) lysates in A549 cells treated with siRNAs for 24 h. siCntr: control scramble siRNAs; siTP53: siRNA specific for TP53; and siAGO2: two different siRNAs specific for AGO2. GAPDH served as a cytoplasmic marker and HIST3H3 served as nuclear marker. n = 3.

Article Snippet: siRNA targeting human AGO2 (siRNA ID: s109013 and ID s25931), TP53 (siRNA ID s607 and S605), TNRC6 (siRNA ID: s26154), and scramble control siRNAs (siRNA ID: 4 390 843) were purchased from ThermoFisher Scientific.

Techniques: Western Blot, Transfection, Control, Expressing, Mutagenesis, Marker, Generated

Journal: Nucleic Acids Research

Article Title: Nuclear AGO2 supports influenza A virus replication through type-I interferon regulation

doi: 10.1093/nar/gkaf268

Figure Lengend Snippet: Nuclear AGO2 downregulates IFNB and other type-I-IFN related genes ( A ) Relative expression, as measured by RT-qPCR, of IFNB mRNA levels in HEK293T and HEK293 cells. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗ P < 0.1 by unpaired t-test. n = 3 ( B ) Relative expression, as measured by RT-qPCR, of IFNB mRNA levels in HEK293 cells infected with PR8 virus at MOI 10 for 2, 8, or 16 h. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗ P < 0.05, ∗∗ P < 0.01 by unpaired t-test. n = 3 ( C ) Representative AGO2 immunoblots from cytoplasmic (C) and nuclear (N) lysates in HEK293 cells infected with PR8 virus at MOI 10 for 2, 8, or 16 h. GAPDH served as a cytoplasmic marker and HIST3H3 served as nuclear marker. n = 3 ( D ) Relative expression, as measured by RT-qPCR, of IFNB mRNA levels in A549 cells treated with 0.5 μg/mL arsenic trioxide (ATO) for 2, 8, or 16 h. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗∗ P < 0.01 by unpaired t-test. n = 3 ( E ) Relative expression, as measured by RT-qPCR, of HA and NS1 mRNA levels in A549 cells treated for 2 h with 0.5 μg/ml arsenic trioxide (ATO)) or vehicle and infected with PR8 virus at MOI 2 or MOI 10 for 16 h. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗ P < 0.05 by unpaired t-test. n = 3 ( F ) same as in (E) but IFNB mRNA level were measured by RT-qPCR. n = 3 ( G ) Relative expression, as measured by RT-qPCR, of IFNB mRNA levels in A549 cells treated with siRNAs against TP53 or AGO2 for 48 h. Sixteen hours before the end of incubation, cells were infected with PR8 virus at MOI 2 or MOI 10. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001 by unpaired t-test. n = 3 ( H ) Relative expression, as measured by RT-qPCR, of IFNAR2 mRNA levels in HEK293 cells treated with siRNA against AGO2 for 48 h. Sixteen hours before the end of incubation, cells were infected with PR8 virus at MOI 2 or MOI 10. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗ P < 0.05 by unpaired t-test. n = 3 ( I ) Normalized lucifersase signal of ISRE-transfected HEK293 cells, treated with siRNA against AGO2 for 48 h. Sixteen hours before the end of incubation, cells were infected with PR8 virus at MOI 10. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗ P < 0.05 by unpaired t-test. n = 3 ( J ) same as in (I) but in A549 cells. n = 3 ( K ) Relative expression, as measured by RT-qPCR, of ISG15, ISG20, OAS1, OAS3, PARRP12, and TRIM25 mRNA levels A549 cells treated with siRNA against AGO2 for 48 h. Sixteen hours before the end of incubation, cells were infected with PR8 virus at MOI 10. GAPDH was used as a reference gene. Bars are mean and error bars represent ± SD. ∗ P < 0.05 by unpaired t-test. n = 3.

Article Snippet: siRNA targeting human AGO2 (siRNA ID: s109013 and ID s25931), TP53 (siRNA ID s607 and S605), TNRC6 (siRNA ID: s26154), and scramble control siRNAs (siRNA ID: 4 390 843) were purchased from ThermoFisher Scientific.

Techniques: Expressing, Quantitative RT-PCR, Infection, Virus, Western Blot, Marker, Incubation, Transfection