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