Topoisomerase III-β is required for efficient replication of positive-sense RNA viruses
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SciScore for 10.1101/2020.03.24.005900: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies: Rabbit monoclonal antibody against TDRD3 was obtained from CST (5942S). TDRD3suggested: (Cell Signaling Technology Cat# 5942, RRID:AB_2797626)Rabbit monoclonal antibody against TOP3B was obtained from ABcam (ab183520). Rabbit monoclonal antibody against TOP3Bsuggested: Noneantibody against TOP3Bsuggested: NoneRabbit monoclonal antibodies against GFP were obtained from ThermoFisher Scientific (G10362). GFPsuggested: (Thermo Fisher Scientific Cat# …SciScore for 10.1101/2020.03.24.005900: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies: Rabbit monoclonal antibody against TDRD3 was obtained from CST (5942S). TDRD3suggested: (Cell Signaling Technology Cat# 5942, RRID:AB_2797626)Rabbit monoclonal antibody against TOP3B was obtained from ABcam (ab183520). Rabbit monoclonal antibody against TOP3Bsuggested: Noneantibody against TOP3Bsuggested: NoneRabbit monoclonal antibodies against GFP were obtained from ThermoFisher Scientific (G10362). GFPsuggested: (Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526)G10362suggested: (Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526)Mouse monoclonal antibodies against Actin were obtained from Santa Cruz Biotechnology (sc-47778). Actinsuggested: (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189)4G2 anti-Flavivirus envelope Mouse monoclonal antibody was isolated from the DI-4G2-4-15 hybridoma, ATCC. anti-Flavivirus envelope Mousesuggested: (Thermo Fisher Scientific Cat# MA1-71258, RRID:AB_962081)Monoclonal Mouse Anti-Enterovirus (clone 5-D8/1) antibody was purchased from Agilent Dako (Code M7064). Anti-Enterovirussuggested: (Agilent Cat# M7064, RRID:AB_2118128)The blots were then washed and incubated with anti-rabbit or anti-mouse fluorophore/DyLight FW 800-conjugated secondary antibody (Santa Cruz Biotechnology). anti-rabbitsuggested: Noneanti-mousesuggested: None72 hpi cells were stained with 4G2 mAb (YFV) or VP1 (CVB3) primary antibody, Alexa647-conjugated anti-mouse IgG mAb as a secondary antibody and DAPI, and the infection rate was obtained from the average of the fthree areas and shown as % infected cells. VP1suggested: NoneBriefly, the cells were stained with 4G2 mAb as a primary antibody, Alexa647-conjugated anti-mouse IgG mAb as a secondary antibody and DAPI. anti-mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Cells, and Viruses: Human hepatoma cells (HuH-7) and African green monkey kidney (Vero) cells were kindly provided by Charles Rice (Rockefeller University), and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2. Verosuggested: NoneHuman Embryonic kidney 293 with FLP-In site (HEK293 Flp-In T-REx) cells were purchased from Thermo Fisher Scientific, and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, zeocin (100 μg/ml) and blasticidin (15 μg/ml) at 37°C with 5% CO2. HEK293suggested: NoneDengue virus-2 (DENV-2 New Guinea C), yellow fever virus (YFV 17D) or Coxsackie virus B3 (CVB3 Strain 20) were prepared as working stock after the virus propagation in C6/36 (DENV-2, YFV) or HeLa (CVB3), respectively 3. HeLasuggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)Virus titration: To determine virus titer, plaque-forming assays of DENV were performed by using BHK21 cells. BHK21suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)Generation of TDRD3 and TOP3B knockout cells by the CRISPR/Cas9 system: To generate TDRD3 KO cells, HuH-7 cells were co-transfected with pX330-TDRD3 gRNA1, pX330-TDRD3 gRNA2, and puromycin plasmid. HuH-7suggested: NoneHuH-7 TOP3B KO cells were generated in the same manner. TOP3Bsuggested: NoneImmunoblotting: The HuH-7 wild-type (WT), HuH-7 TDRD3 KO, or HuH-7 TOP3B KO cells were lysed in RIPA buffer (Cell Signaling Technologies) with 1X protease inhibitor cocktail (Cell Signaling Technologies) and centrifuged at 10,000 rpm for 3 min to pellet membranous cell debris. HuH-7 TOP3B KOsuggested: NoneIn brief, for plaque assays, confluent monolayers of MDCK cells were washed once with DPBS. MDCKsuggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)Experimental Models: Organisms/Strains Sentences Resources Immunoblotting: The HuH-7 wild-type (WT), HuH-7 TDRD3 KO, or HuH-7 TOP3B KO cells were lysed in RIPA buffer (Cell Signaling Technologies) with 1X protease inhibitor cocktail (Cell Signaling Technologies) and centrifuged at 10,000 rpm for 3 min to pellet membranous cell debris. HuH-7suggested: NoneSoftware and Algorithms Sentences Resources Statistical analyses were conducted in Prism8 (GraphPad Software). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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