Journal: Cell reports

Article Title: Alphavirus infection triggers antiviral RNAi immunity in mammals.

doi: 10.1016/j.celrep.2023.112441

Figure Lengend Snippet: Figure 2. The production of SFV vsiRNAs is Dicer dependent Normal 293T cells, AGO2-KO 293T cells, or NoDice 293T cells were infected with SFV (MOI = 1). RNA extracts were subjected to small RNA sequencing at 24 h.p.i. (n = 3 replicates).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-pan AGO clone 2A8 EMD MILLIPORE Cat# MABE56; RRID: AB_11214388 Rabbit monoclonal anti-AGO2 (EPR10410) Abcam Cat# ab156870; RRID: AB_2687492 Mouse monoclonal anti-Dicer (13D6) Abcam Cat# ab14601; RRID: AB_443067 Rabbit monoclonal anti-RNase L (EPR15894) Abcam Cat# ab191392; RRID: AB_2916269 Neurofilament-L (DA2) Mouse mAb Cell Signaling Technology Cat# 2835; RRID: AB_490808 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664; RRID: AB_2070042 Mouse anti-dsRNA monoclonal antibody J2 Scicons Cat# 10010200; RRID: AB_2922431 Mouse IgG ProteinTech Cat# B900620 Anti-DDDDK-tag mAb-HRP-DirecT MBL Cat# M185-7 HRP-conjugated GAPDH Mouse mAb Abclonal Cat# AC035 HRP-conjugated Alpha Tubulin Monoclonal antibody ProteinTech Cat#HRP-66031 Bacterial and virus strains SFV Qian et al., 202030 N/A SFVK124A/K128A Qian et al., 2020 30 N/A SFVmutAA This paper N/A SINV Qian et al., 202030 N/A Chemicals, peptides, and recombinant proteins Protein A/G Magnetic Beads MCE Cat# HY-K0202 Enoxacin Sigma Cat# 557305 Cell Counting Kit-8 (CCK-8) Dojindo Cat# CK04 RNAiso Plus Takara Cat# 9108 Cocktail Targetmol Cat# C0001 ShortCut RNase III NEB Cat# M0245S Critical commercial assays Cell Total RNA Isolation Kit Foregene Cat# RE-03113 Hieff qPCR SYBR Green Master Mix Yeasen Cat# 11202ES03 TruSeq Small RNA Library Preparation Kit Llumina Cat# RS-200-0012 Deposited data RNA-seq data This paper GEO: GSE214453 Mass spectrometry proteomics data This paper PXD030194 Experimental models: Cell lines HEK293T cells ATCC Cat# CRL-11268; RRID: CVCL_1926 293T NoDice cells Bogerd et al., 201445 N/A 293T AGO2-KO cells This paper N/A 293T NoDice/RNase L-KO cells This paper N/A BHK-21 cells ATCC Cat# CCL-10; RRID: CVCL_1915 Vero cells ATCC Cat# CCL-81; RRID: CVCL_0059 Experimental models: Organisms/strains C57BL/6 mice Zhang et al., 201646 N/A Ifnar1-/- C57BL/6 mice Zhang et al., 201646 N/A Oligonucleotides Oligonucleotides, see Table S5 This paper N/A Primers for qRT-PCR, see Table S5 This paper N/A (Continued on next page) 16 Cell Reports 42, 112441, May 30, 2023

Techniques: Infection, RNA Sequencing

Journal: Cell reports

Article Title: Alphavirus infection triggers antiviral RNAi immunity in mammals.

doi: 10.1016/j.celrep.2023.112441

Figure Lengend Snippet: Figure 3. SFV vsiRNAs silence cognate viral RNA sequence and confer effective antiviral activity in human 293T cells (A) Schematic diagram of the plasmid containing the egfp ORF followed by the 1–50 nt (pEGFP-SFV1–50) or 451–500 nt (pEGFP-SFV451–500) of SFV genome (top). Normal or NoDice 293T cells were transfected with the indicated plasmids (0.5 mg), and after 4-h transfection, cells were infected with SFV (MOI = 0.1). At 12 h.p.i., total RNAs were subjected to qRT-PCR to detect EGFP mRNA (n = 3 replicates), and the level of EGFP mRNA in uninfected cells (none) expressing pEGFP451–500 was defined as 100%. (B) Normal or AGO2-KO 293T cells were infected with SFV (MOI = 0.1), and at 4, 8, and 12 h.p.i., the levels of SFV RNA were determined via qRT-PCR (n = 3 replicates). The level of SFV RNA in infected 293T cells at 4 h.p.i. was defined as 1-fold. The viral titers in the supernatant were measured (n = 4 replicates). (C) Normal or AGO2-KO 293T cells expressing the vector (control), human AGO2WT, or AGO2D597A were infected with SFV (MOI = 0.1). At 12 h.p.i., SFV RNAs were determined via qRT-PCR (n = 4 replicates), and the level of SFV RNA in infected control cells (293T + Vector) at 12 h.p.i. was defined as 100%.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-pan AGO clone 2A8 EMD MILLIPORE Cat# MABE56; RRID: AB_11214388 Rabbit monoclonal anti-AGO2 (EPR10410) Abcam Cat# ab156870; RRID: AB_2687492 Mouse monoclonal anti-Dicer (13D6) Abcam Cat# ab14601; RRID: AB_443067 Rabbit monoclonal anti-RNase L (EPR15894) Abcam Cat# ab191392; RRID: AB_2916269 Neurofilament-L (DA2) Mouse mAb Cell Signaling Technology Cat# 2835; RRID: AB_490808 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664; RRID: AB_2070042 Mouse anti-dsRNA monoclonal antibody J2 Scicons Cat# 10010200; RRID: AB_2922431 Mouse IgG ProteinTech Cat# B900620 Anti-DDDDK-tag mAb-HRP-DirecT MBL Cat# M185-7 HRP-conjugated GAPDH Mouse mAb Abclonal Cat# AC035 HRP-conjugated Alpha Tubulin Monoclonal antibody ProteinTech Cat#HRP-66031 Bacterial and virus strains SFV Qian et al., 202030 N/A SFVK124A/K128A Qian et al., 2020 30 N/A SFVmutAA This paper N/A SINV Qian et al., 202030 N/A Chemicals, peptides, and recombinant proteins Protein A/G Magnetic Beads MCE Cat# HY-K0202 Enoxacin Sigma Cat# 557305 Cell Counting Kit-8 (CCK-8) Dojindo Cat# CK04 RNAiso Plus Takara Cat# 9108 Cocktail Targetmol Cat# C0001 ShortCut RNase III NEB Cat# M0245S Critical commercial assays Cell Total RNA Isolation Kit Foregene Cat# RE-03113 Hieff qPCR SYBR Green Master Mix Yeasen Cat# 11202ES03 TruSeq Small RNA Library Preparation Kit Llumina Cat# RS-200-0012 Deposited data RNA-seq data This paper GEO: GSE214453 Mass spectrometry proteomics data This paper PXD030194 Experimental models: Cell lines HEK293T cells ATCC Cat# CRL-11268; RRID: CVCL_1926 293T NoDice cells Bogerd et al., 201445 N/A 293T AGO2-KO cells This paper N/A 293T NoDice/RNase L-KO cells This paper N/A BHK-21 cells ATCC Cat# CCL-10; RRID: CVCL_1915 Vero cells ATCC Cat# CCL-81; RRID: CVCL_0059 Experimental models: Organisms/strains C57BL/6 mice Zhang et al., 201646 N/A Ifnar1-/- C57BL/6 mice Zhang et al., 201646 N/A Oligonucleotides Oligonucleotides, see Table S5 This paper N/A Primers for qRT-PCR, see Table S5 This paper N/A (Continued on next page) 16 Cell Reports 42, 112441, May 30, 2023

Techniques: Sequencing, Activity Assay, Plasmid Preparation, Transfection, Infection, Quantitative RT-PCR, Expressing, Control

Journal: Cell reports

Article Title: Alphavirus infection triggers antiviral RNAi immunity in mammals.

doi: 10.1016/j.celrep.2023.112441

Figure Lengend Snippet: Figure 4. The conserved sequence at the 50 terminus of SFV genome is required for vsiRNA production (A) Schematic illustration of SFV genome and the mutation sites (SFVmutAA) in the 50 UTR. (B) The plaque morphology of SFVWT and SFVmutAA in Vero cells. (C) 293T cells were infected with SFVWT or SFVmutAA (MOI = 0.1). Viral titers were measured (n = 4 replicates). (D–H) MLFs were infected with SFVmutAA (MOI = 10), and total RNAs were subjected to small RNA sequencing at 24 h.p.i. (n = 3 replicates). (D) Size distribution and abundance (CPM) of total vsRNAs. (E) Frequency of each distance category between 50 and 30 ends of a 22-nt vsRNA pair. (F) The distribution and abundance of

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-pan AGO clone 2A8 EMD MILLIPORE Cat# MABE56; RRID: AB_11214388 Rabbit monoclonal anti-AGO2 (EPR10410) Abcam Cat# ab156870; RRID: AB_2687492 Mouse monoclonal anti-Dicer (13D6) Abcam Cat# ab14601; RRID: AB_443067 Rabbit monoclonal anti-RNase L (EPR15894) Abcam Cat# ab191392; RRID: AB_2916269 Neurofilament-L (DA2) Mouse mAb Cell Signaling Technology Cat# 2835; RRID: AB_490808 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664; RRID: AB_2070042 Mouse anti-dsRNA monoclonal antibody J2 Scicons Cat# 10010200; RRID: AB_2922431 Mouse IgG ProteinTech Cat# B900620 Anti-DDDDK-tag mAb-HRP-DirecT MBL Cat# M185-7 HRP-conjugated GAPDH Mouse mAb Abclonal Cat# AC035 HRP-conjugated Alpha Tubulin Monoclonal antibody ProteinTech Cat#HRP-66031 Bacterial and virus strains SFV Qian et al., 202030 N/A SFVK124A/K128A Qian et al., 2020 30 N/A SFVmutAA This paper N/A SINV Qian et al., 202030 N/A Chemicals, peptides, and recombinant proteins Protein A/G Magnetic Beads MCE Cat# HY-K0202 Enoxacin Sigma Cat# 557305 Cell Counting Kit-8 (CCK-8) Dojindo Cat# CK04 RNAiso Plus Takara Cat# 9108 Cocktail Targetmol Cat# C0001 ShortCut RNase III NEB Cat# M0245S Critical commercial assays Cell Total RNA Isolation Kit Foregene Cat# RE-03113 Hieff qPCR SYBR Green Master Mix Yeasen Cat# 11202ES03 TruSeq Small RNA Library Preparation Kit Llumina Cat# RS-200-0012 Deposited data RNA-seq data This paper GEO: GSE214453 Mass spectrometry proteomics data This paper PXD030194 Experimental models: Cell lines HEK293T cells ATCC Cat# CRL-11268; RRID: CVCL_1926 293T NoDice cells Bogerd et al., 201445 N/A 293T AGO2-KO cells This paper N/A 293T NoDice/RNase L-KO cells This paper N/A BHK-21 cells ATCC Cat# CCL-10; RRID: CVCL_1915 Vero cells ATCC Cat# CCL-81; RRID: CVCL_0059 Experimental models: Organisms/strains C57BL/6 mice Zhang et al., 201646 N/A Ifnar1-/- C57BL/6 mice Zhang et al., 201646 N/A Oligonucleotides Oligonucleotides, see Table S5 This paper N/A Primers for qRT-PCR, see Table S5 This paper N/A (Continued on next page) 16 Cell Reports 42, 112441, May 30, 2023

Techniques: Sequencing, Mutagenesis, Infection, RNA Sequencing

Journal: Cell reports

Article Title: Alphavirus infection triggers antiviral RNAi immunity in mammals.

doi: 10.1016/j.celrep.2023.112441

Figure Lengend Snippet: Figure 2. The production of SFV vsiRNAs is Dicer dependent Normal 293T cells, AGO2-KO 293T cells, or NoDice 293T cells were infected with SFV (MOI = 1). RNA extracts were subjected to small RNA sequencing at 24 h.p.i. (n = 3 replicates).

Article Snippet: The Ago2 CRISPR/Cas9 KO Plasmid (sc-400813, Santa Cruz Biotech) was used to knockout ago2 gene in 293T cells as described previously.47 Briefly, after transfection with the CRISPR/Cas9 KO plasmids, 293T cells were cultured for 3 days and selected by puromycin (1 mg/mL) (Invivogen).

Techniques: Infection, RNA Sequencing

Journal: Cell reports

Article Title: Alphavirus infection triggers antiviral RNAi immunity in mammals.

doi: 10.1016/j.celrep.2023.112441

Figure Lengend Snippet: Figure 3. SFV vsiRNAs silence cognate viral RNA sequence and confer effective antiviral activity in human 293T cells (A) Schematic diagram of the plasmid containing the egfp ORF followed by the 1–50 nt (pEGFP-SFV1–50) or 451–500 nt (pEGFP-SFV451–500) of SFV genome (top). Normal or NoDice 293T cells were transfected with the indicated plasmids (0.5 mg), and after 4-h transfection, cells were infected with SFV (MOI = 0.1). At 12 h.p.i., total RNAs were subjected to qRT-PCR to detect EGFP mRNA (n = 3 replicates), and the level of EGFP mRNA in uninfected cells (none) expressing pEGFP451–500 was defined as 100%. (B) Normal or AGO2-KO 293T cells were infected with SFV (MOI = 0.1), and at 4, 8, and 12 h.p.i., the levels of SFV RNA were determined via qRT-PCR (n = 3 replicates). The level of SFV RNA in infected 293T cells at 4 h.p.i. was defined as 1-fold. The viral titers in the supernatant were measured (n = 4 replicates). (C) Normal or AGO2-KO 293T cells expressing the vector (control), human AGO2WT, or AGO2D597A were infected with SFV (MOI = 0.1). At 12 h.p.i., SFV RNAs were determined via qRT-PCR (n = 4 replicates), and the level of SFV RNA in infected control cells (293T + Vector) at 12 h.p.i. was defined as 100%.

Article Snippet: The Ago2 CRISPR/Cas9 KO Plasmid (sc-400813, Santa Cruz Biotech) was used to knockout ago2 gene in 293T cells as described previously.47 Briefly, after transfection with the CRISPR/Cas9 KO plasmids, 293T cells were cultured for 3 days and selected by puromycin (1 mg/mL) (Invivogen).

Techniques: Sequencing, Activity Assay, Plasmid Preparation, Transfection, Infection, Quantitative RT-PCR, Expressing, Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: Accumulation of Ago2 to SGs is inhibited by shRNA transfection. RPTC cells were treated with or without arsenite for 45 min. Cells were fixed and stained for Ago2 and eIF3η. A: representative confocal microscopy images of cells (scale bar = 10 μm). B: co-IP assay. Hsf1 shRNA knockdown cells and negative control cells were treated with or without arsenite for 45 min. Cell lysate was then collected for co-IP with anti-Ago2 antibody followed by immunoblotting of eIF3η, HuR, PKCα, and Ago2. C: HEK293 cells were transfected with Flag-Ago2 plasmid and then treated with or without arsenite for 45 min. The cell lysates were immunoprecipitated with Flag antibody and immunoblotted for eIF3, TIA1, HuR, and Ago2. Ago2, argonaute 2; co-IP, coimmunoprecipitation; eIF3, eukaryotic initiation factor 3; Hsf1, heat shock transcription factor 1; HEK293, human embryonic kidney 293; HuR, human antigen R; KD, knockdown; NC, negative control; RPTC, renal proximal tubule cells; SG, stress granules; TIA1, T-cell intracellular antigen 1.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: shRNA, Transfection, Staining, Confocal Microscopy, Co-Immunoprecipitation Assay, Negative Control, Western Blot, Plasmid Preparation, Immunoprecipitation

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: Kinetic interaction of Ago2 with RISC marker TRBP2 during arsenite treatment. A: co-IP assay. Hsf1 shRNA knockdown and negative control RPTC cells were treated with arsenite for 0, 15, 30, 45, or 60 min. Cell lysate was collected for co-IP with anti-TRBP2 antibody followed by immunoblotting of Ago2 and TRBP2. B: kinetic modeling of Ago2 dissociation from TRBP2 in Hsf1 KD cells and NC cells. The changes of TRBP2-associated Ago2 during arsenite treatment were semiquantified by densitometry. Ago2, argonaute 2; co-IP, coimmunoprecipitation; Hsf1, heat shock transcription factor 1; KD, knockdown; NC, negative control; RISC, RNA-induced silencing complex; RPTC, renal proximal tubule cells; TRBP2, trans-activation-responsive RNA-binding protein.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: Marker, Co-Immunoprecipitation Assay, shRNA, Negative Control, Western Blot, Activation Assay, RNA Binding Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: SG formation is reduced in Ago2-deficient cells. A: schematic diagram of CRISPR-mediated Ago2-GFP/Puro recombination. G1 and G2 represent two guide RNA sequences in the chromosome that guide caspase-9 enzyme to cut its adjacent downstream sites. Donor vector contained 600-bp-long 5′- and 599-bp-long 3′-homologous arms flanking the gene of interest (2,588 bp). LHA, left homologous arm; RHA, right homologous arm. B: Western blot analysis of Ago2 expression in two Ago2−/− clones (#1, #2). Cyclophilin B served as a loading control. C: Ago2 knockout cells (Ago2 KO) and wild-type cells (WT) were treated with arsenite for 45 min. Cells were immediately fixed for eIF3η immunofluorescence. D: percentage of cells with SGs as calculated by 100 × [(number of cells with SGs)/(total number of cells)]. Data are presented as means ± SE (n = 3). *P < 0.05 vs. NC in Student’s t-test. Ago2, argonaute 2; eIF3, eukaryotic initiation factor 3; GFP, green fluorescent protein; NC, negative control; SG, stress granule.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: CRISPR, Plasmid Preparation, Western Blot, Expressing, Clone Assay, Knock-Out, Immunofluorescence, Negative Control

Journal: American Journal of Physiology - Cell Physiology

Article Title: RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment

doi: 10.1152/ajpcell.00251.2018

Figure Lengend Snippet: Schematic diagram of the competing relationship between stress granules (SG) and RNA-induced silencing complex (RISC) for key protein components such as (Ago2). HuR, human antigen R; eIF3, eukaryotic initiation factor 3.

Article Snippet: Ago2 KO was performed by using the CRISPR/Cas9 system (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"KN218078","term_id":"694262471","term_text":"KN218078"}} KN218078 ; Origene, Rockville, MD).

Techniques: