Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: ZIKV sfRNA inhibits shRNA-induced RNAi in hNPCs. (A) hNPCs were cotransfected with plasmids pEGFP-N1 (encoding eGFP) (100 ng), shGFP, or shNC (300 ng), and ZIKV sfRNA or control RNA (300 ng). The expression of eGFP was analyzed 48 h after cotransfection. The intensity of eGFP was observed under fluorescence microscopy. (B) Cell lysates from (A) were harvested and analyzed by Western blot. (C) The levels of GFP expression in panel A of three independent experiments were quantified by densitometry and normalized to that of actin. The expression level of GFP in the first column was set at 1.0. (D) hNPCs were infected with ZIKV or △10 ZIKV. Thirty-six hours post-infection, cell-associated RNA was harvested and levels of gRNA and sfRNA were analyzed by Northern Blot. (E) hNPCs were mock infected or infected with ZIKV FSS13025 (MOI = 2) or △10 ZIKV(MOI = 5) (different MOIs were used to achieve similar infection rates). Twelve hours after infection, cells were cotransfected with plasmids as indicated. The expression of eGFP was analyzed 36 h after cotransfection. The intensity of eGFP was observed under fluorescence microscopy. (F) Cell lysates from (D) were harvested and analyzed by Western blot. (G) The levels of GFP expression in panel F of three independent experiments were quantified by densitometry and normalized to that of actin. The expression level of GFP in the first column was set at 1.0. Scale bar, 200 μm. Data are expressed as mean ± SD from three independent experiments. *P ≤ 0.05, ***P ≤ 0.001, as determined by Student’s t test.

Article Snippet: 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: shRNA, Control, Expressing, Cotransfection, Fluorescence, Microscopy, Western Blot, Infection, Northern Blot

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: ZIKV sfRNA competes with siRNA for binding to RHA and PACT. (A) SDS-PAGE of purified recombinant RHA (tandem dsRBDs, 1–264aa) and PACT. (B) Increasing amounts of unlabeled ZIKV sfRNA [left panel: 0.5 (lane 3), 2 (lane 4), 9 (lane5), or 27 (lane 6) pmoles; right panel: 0.2 (lane 3), 0.5 (lane 4), 1 (lane5), or 3 (lane 6) pmoles] (B) or control RNA (C) were incubated with 4 µM protein and 10 nM biotin-labeled siRNA as indicated in a 20 μL reaction. Samples were separated on a 6% native-TBE polyacrylamide gel electrophoresis (PAGE), transferred to membranes, and then incubated with Horseradish-conjugated Streptavidin.

Article Snippet: 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Binding Assay, SDS Page, Purification, Recombinant, Control, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: ZIKV sfRNA antagonizes antiviral function of RNAi. hNPCs were transfected with control siRNA (siCtrl) and siRNAs targeting RHA (siRHA) or PACT(siPACT). Thirty-six hours after transfection, cells were infected with ZIKV FSS13025 at MOI = 1 for 36 h. (A) Representative Western blot of RHA or PACT knockdown efficiency. (B) Viral RNA copies in supernatants were measured by RT-qPCR. (C) Fold change of (B). (D) The expression of viral envelope protein from (B) was detected by immunostaining. Viral infection rates were measured by envelope protein signal. Scale bar, 50 μm. Data are expressed as mean ± SD from three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, as determined by Student’s t test.

Article Snippet: 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Transfection, Control, Infection, Western Blot, Knockdown, Quantitative RT-PCR, Expressing, Immunostaining

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: ZIKV 3′SL rescues the replication of VSR-deficient HEV71. (A and B) 293T cells were transfected with control siRNA (siCtrl) and siRNAs targeting RHA (siRHA) and PACT (siPACT). Twenty-four hours after transfection, cells were transfected with Ctrl RNA or ZIKV 3′SL RNA. Six hours after transfection, cells were infected with HEV71D23A at an MOI = 0.01 as indicated. At 24–72 hpi, the levels of HEV71D23A genomic RNAs were determined via RT-qPCR. The level of HEV71D23A RNA in cells at 24 hpi was defined as 1 (A). The viral titers at 72 hpi were determined (B). (C) The RHA and PACT knockdown efficiency was detected by Western blot. Data are expressed as mean ± SD from three independent experiments. **P ≤ 0.01, ***P ≤ 0.001, as determined by Student’s t test.

Article Snippet: 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Transfection, Control, Infection, Quantitative RT-PCR, Knockdown, Western Blot

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: Multiple flaviviral 3′SLs can bind to RISC components and exhibit higher affinities than siRNA. (A) Biotinylated 3′SL RNAs from ZIKV, DENV, WNV, JEV, YFV, or siRNA were incubated with hNPC lysates. Associated proteins were eluted, separated on a 10% SDS-PAGE and probed with the antibodies indicated on the left by Western blot. (B) BLI assay. Biotinylated RNAs were captured onto streptavidin (SA) biosensors and assayed for binding to RHA or PACT at the indicated concentrations. The data collected were processed on the Gator software. (C) The binding affinities (KD) of (B) were compared.

Article Snippet: 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Incubation, SDS Page, Western Blot, Binding Assay, Software

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: Model for the suppression of antiviral RNAi by flavivirus. During flavivirus infection, the virus-derived dsRNA is cleaved by Dicer into siRNAs, and then the dsRBPs (RHA and PACT) bind and help to reposition siRNAs for loading them onto Ago. Finally, the mature RISC forms and actives RNAi for viral RNA degradation. However, the viral proteins capsid and NS2A and sfRNA can inhibit Dicer from cleaving dsRNA to siRNA. The abundant sfRNA can also compete with siRNA for binding to RHA and PACT through its 3′SL, leading to the suppression of active RISC assembly and subsequent target cleavage.

Article Snippet: 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Infection, Virus, Derivative Assay, Binding Assay

Journal: Journal of Virology

Article Title: The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components

doi: 10.1128/jvi.01954-23

Figure Lengend Snippet: ZIKV sfRNA competes with siRNA for binding to RHA and PACT. (A) SDS-PAGE of purified recombinant RHA (tandem dsRBDs, 1–264aa) and PACT. (B) Increasing amounts of unlabeled ZIKV sfRNA [left panel: 0.5 (lane 3), 2 (lane 4), 9 (lane5), or 27 (lane 6) pmoles; right panel: 0.2 (lane 3), 0.5 (lane 4), 1 (lane5), or 3 (lane 6) pmoles] (B) or control RNA (C) were incubated with 4 µM protein and 10 nM biotin-labeled siRNA as indicated in a 20 μL reaction. Samples were separated on a 6% native-TBE polyacrylamide gel electrophoresis (PAGE), transferred to membranes, and then incubated with Horseradish-conjugated Streptavidin.

Article Snippet: Blots were developed using an enhanced chemiluminescence (ECL) kit. . Sirna transfection 30 pmoles RHA siRNA (sc-45706 santa cruz) or PACT siRNA (sc-36175 santa cruz) or control siRNA (sc-37007 santa cruz) per well was transfected in a 24-well plate using RNAiMAX reagent (Thermo Fisher Scientific) as recommended by the manufacturer.

Techniques: Binding Assay, SDS Page, Purification, Recombinant, Control, Incubation, Labeling, Polyacrylamide Gel Electrophoresis

Journal: Oncotarget

Article Title: DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels

doi: 10.18632/oncotarget.25104

Figure Lengend Snippet: ( A ) HepG2 cells were transfected with HBV minicircle construct (mc). Three days after transfection, RNAs were extracted and subjected to complementary DNA (cDNA) synthesis by reverse transcription using oligo-dT (Oligo-dT) or random (Random) primers as indicated. Cells without transfection were used as negative controls (NC). PCR was performed using the indicated primer sets. Primer set #1 was used to amplify the core region, and set #2 and set #3 were outward facing primers, which would theoretically result in no amplification. Mcl1 amplification was used as an internal control. RNAs that were not reverse transcribed (RT(−)) were included as negative controls for the PCR to confirm no DNA contamination. Mr, DNA marker. A representative image of two independent experiments is shown. ( B ) RNAs extracted from HepG2 cells transfected with HBV minicircle construct (mc) for 3 days were treated with RNase R and subjected to RT-PCR using random primers and primer set #2. Untransfected cells were used as a negative control (NC). ( C ) HepG2 cells were transfected with HBV pgRNA expressing plasmid (pg). Two days after transfection, extracted RNA was treated with or without RNase R and reverse transcribed using random primers. PCR was performed using primer set #2 and primers for Mcl1 gene amplification as a control. RNAs that were not reverse transcribed (RT(−)) were used as negative controls. Mr, DNA marker. A representative image of two independent experiments is shown. ( D ) DHX9 knockdown leads to increased viral circular RNA production. HepAD38 cells with HBV shut off (Off), HBV continuous expression (On), and HBV continuous expression with shDHX9 expression (On + shDHX9) were subjected to northern blotting with or without RNase R treatment. Probes against HBV RNA primarily spanning the core region and β-actin were used. Gel staining with ethidium bromide is shown to confirm RNA integrity (bands of 28S and 18S ribosomal RNA) and the effects of RNase treatment (disappearance of ribosomal RNA bands). A representative image of two independent experiments is shown.

Article Snippet: shRNA-expressing lentiviruses against each protein were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): shDNA-PK (sc-35200-V), shsnRNP200 (sc-75243-V), shILF3 (sc-106301-V), shDHX9 (sc-45706-V), and shLRPPRC (sc-44734-V).

Techniques: Transfection, Construct, cDNA Synthesis, Reverse Transcription, Amplification, Control, Marker, Reverse Transcription Polymerase Chain Reaction, Negative Control, Expressing, Plasmid Preparation, Knockdown, Northern Blot, Staining

Journal: Oncotarget

Article Title: DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels

doi: 10.18632/oncotarget.25104

Figure Lengend Snippet: ( A ) A scheme for HBV infection in human primary hepatocytes. At HBV infection, lentivirus-expressing shDHX9 was also applied simultaneously to knock down DHX9. Cells were harvested at 17 days after infection. ( B ) Cell lysates were subjected to western blotting to determine DHX9 and HBV surface protein levels in DHX9-knockdown cells. ( C ) RNAs extracted from the HBV-infected human primary hepatocytes were treated with DNase and subsequently with RNase R, followed by reverse transcription using random primers. cDNAs were subjected to droplet digital PCR using Taqman primers for the absolute quantification of HBV circular DNA. The measurement was performed in triplicate. Data represent the mean ± standard error (SE) of the absolute copy numbers from two independent experiments. * p < 0.05. ( D ) Real-time PCR was performed to measure pgRNA levels (including circular RNA) using the total RNAs from HBV-infected human primary hepatocytes. Following DNase treatment, reverse transcription was performed using random primers, and real-time PCR amplifying the core region was performed in triplicate. The data were normalized with β-actin. Data represent the mean ± SE of two independent experiments. n.s., no significant differences.

Article Snippet: shRNA-expressing lentiviruses against each protein were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): shDNA-PK (sc-35200-V), shsnRNP200 (sc-75243-V), shILF3 (sc-106301-V), shDHX9 (sc-45706-V), and shLRPPRC (sc-44734-V).

Techniques: Infection, Expressing, Knockdown, Western Blot, Reverse Transcription, Digital PCR, Quantitative Proteomics, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: LncRNA AK023948 is a positive regulator of AKT

doi: 10.1038/ncomms14422

Figure Lengend Snippet: ( a ) RNA precipitation using the biotin-labelled AK023948 probe, followed by PAGE and silver staining. Red star indicates a unique band bound to AK023948. Mass spectrometry analysis suggested DHX9 as a candidate. ( b ) Confirmation of the interaction between AK023948 and DHX9 by RNA precipitation and western blot. ( c ) Confirmation of the interaction between AK023948 and DHX9 by RNA immunoprecipitation using DHX9 antibody. ( d ) While DHX9 siRNA suppresses, ectopic expression of DHX9 increases the pAKT level. DHX9 siRNAs or DHX9 expression vector was introduced into MCF-7 cells by transfection, and cellular extract was prepared for western blot 48 h after transfection. Values in c are s.e.m. ( n =3). ** P <0.01 by two-tailed Student's t -test.

Article Snippet: DHX9 siRNAs (#sc-45706, pooled) and p85β siRNAs (#sc-39125, pooled) were purchased from Santa Cruz.

Techniques: Silver Staining, Mass Spectrometry, Western Blot, RNA Immunoprecipitation, Expressing, Plasmid Preparation, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: LncRNA AK023948 is a positive regulator of AKT

doi: 10.1038/ncomms14422

Figure Lengend Snippet: ( a ) AK023948 KO primarily suppresses the p85β level, as detected by western blot. ( b ) AK023948 interacts with p85, as detected by RIP assay using p85 antibody. ( c ) AK023948 is required for the interaction between DHX9 and p85, as detected by co-immunoprecipitation (co-IP) using p85 antibody. HMLE cells express little AK023948, whereas MCF-7 cells express a high level of AK023948. ( d ) Rescue experiments further suggest that AK023948 is critical for the interaction between AK023948 and p85. ( e ) AK023948 is required for the interaction between DHX9 and p85, as determined by GST pulldown assay. The DHX9 level pulled down by GST-p85 was lower in KO cells than in gRNA control. ( f ) AK023948 is required for the interaction between DHX9 and p85, as detected by the Duolink in situ Fluorescence Kit (Sigma). HeLa cells were transfected with Myc-p85 plus control siRNA or AK0 siRNA. After 48 h, the cells were fixed for PLA. The red signals were lower in AK0 siRNA than in control siRNA cells. Scale bar, 100 μm. ( g ) DHX9 siRNAs suppress, but ectopic expression of DHX9 increases both p85 and pAKT. Values in b are s.e.m. ( n =3). * P <0.05 by two-tailed Student's t -test.

Article Snippet: DHX9 siRNAs (#sc-45706, pooled) and p85β siRNAs (#sc-39125, pooled) were purchased from Santa Cruz.

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, GST Pulldown Assay, Control, In Situ, Fluorescence, Transfection, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: LncRNA AK023948 is a positive regulator of AKT

doi: 10.1038/ncomms14422

Figure Lengend Snippet: ( a ) Detection of AK023948 in the OriGene breast cancer tissue cDNA array by qPCR. ( b ) AK023948 is upregulated in breast cancer cells (MCF-7 and MDA-MB-231) as compared with non-malignant breast cells (MCF-10A and HMLE). ( c ) AK023948 KO suppresses cell proliferation of MCF-7, as detected by MTT assay. ( d ) AK023948 KO promotes apoptosis. Cells were seeded in slide chambers, and treated with H 2 O 2 at 0.8 mM for 4 h before TUNEL assay. Apoptotic cells were counted from three different fields, and apoptotic cell ratio was calculated against total cells. ( e ) Tumour growth for vector control and AK0 KO#13 in nude mice. ( f ) Expression of AK023948 in breast cancer TMAs, as detected by ISH. Scale bar, 100 μm. Bottom: quantitative analysis of AK023948 expression based on the results from f . ( g ) Upregulation of DHX9 expression is associated with poor patient survival. About 20.9% (229 of 1091 cases analysed) are positive for DHX9 using the Onco Query Language (OQL; EXP>1.5). Values in b , c are s.e.m. ( n =3). ** P <0.01 by two-tailed Student's t -test.

Article Snippet: DHX9 siRNAs (#sc-45706, pooled) and p85β siRNAs (#sc-39125, pooled) were purchased from Santa Cruz.

Techniques: MTT Assay, TUNEL Assay, Plasmid Preparation, Control, Expressing, Two Tailed Test