Journal: bioRxiv

Article Title: Lentiviral vector-based SARS-CoV-2 pseudovirus enables analysis of neutralizing activity in COVID-19 convalescent plasma

doi: 10.1101/2020.12.28.424590

Figure Lengend Snippet: Upper panels in (A) and (B) represent the maps for the empty LeGO-iT2puro vector backbone and LeGO-hACE2-iT2puro vector used to genetically modify 293FT cells. Lower panels show representative flow cytometry analysis of RBD-GFP fusion protein staining on tdTomato or hACE2-tdTomato expressing 293FT cells. (C) Representative confocal microscopy images generated from RBD-GFP fusion protein stained tdTomato and hACE2-tdTomato expressing HEK293FT cells.

Article Snippet: For preparation of the vector, LeGO-iT2 and LeGO-iT2puro plasmids were cut by NotI-HF (New England Biolabs (NEB), USA) and ligation with the NotI digested PCR products was performed for 1 hour at room temperature with T4 DNA ligase (NEB).

Techniques: Plasmid Preparation, Flow Cytometry, Staining, Expressing, Confocal Microscopy, Generated

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: Cell cycle arrest gene targets of miR-642a-5p. ( a ) Ingenuity Pathway Analysis of cell cycle targets of miR-642a-5p. Green denotes genes downregulated by miR-642a-5p, and the number of seed sites in their 3’UTR (identified by TargetScan 7.2) indicated. Red denotes genes upregulated by miR-642a-5p. ( b ) RT-qPCR analysis of the cell cycle genes following overexpression of miR-642a-5p in 22Rv1 PCa cells. Expression of target mRNAs is normalized to HPRT housekeeping gene expression, calculated using the 2 −ΔΔCt method, and relative to miR-NC. Error bars = SE; n = 3; * p < 0.05 relative to miR-NC. ( c ) Oncomine analysis of the expression of the miR-642a-5p targets in PCa data sets. (i) WT1; (ii) NUAK1; (iii) RASSF3; (iv) SKP2; (v) IGFBP3; and (vi) GPS2. The data cohorts indicated above each graph, and n per group shown. Boxes denote the median (horizontal line); whiskers indicate distances to the highest and lowest values [for NUAK1 and RASSF3 the lower whisker is to the 10th percentile (minimum value removed)]. * p < 0.05, ** p < 0.005.

Article Snippet: Human WT1 cDNA (WT1-203, ENST00000379079.8, Ensembl) was synthesized (GenScript) and cloned into LeGO-iT2 lentiviral vector (a gift from Boris Fehse, Addgene plasmid # 27343).

Techniques: Quantitative RT-PCR, Over Expression, Expressing, Gene Expression, Whisker Assay

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: WT1 is a direct target of miR-642a-5p in PCa cells. ( a ) The 3′UTR of WT1 has three putative miR-642a-5p seed sites as predicted by TargetScan 7.2. ( b ) Schematic of the 3′UTR of WT1 (not to scale). Depiction of the GeneCopoeia target clone, which contains only the first 1293 base pairs of the 3′UTR, is with green shading. The grey shaded boxes indicate the miR-642a-5p seed sites. ( c ) Luciferase reporter gene analysis of the 3′UTR of the putative miR-642a-5p target WT1 in 22Rv1 and LNCaP PCa cells transiently overexpressing miR-642a-5p or miR-NC (20 nM). DOHH and miR-642a-5p perfect targets are positive controls. Error bars = SD; n = 3; ** p < 0.005.

Article Snippet: Human WT1 cDNA (WT1-203, ENST00000379079.8, Ensembl) was synthesized (GenScript) and cloned into LeGO-iT2 lentiviral vector (a gift from Boris Fehse, Addgene plasmid # 27343).

Techniques: Luciferase

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: Targeted siRNA-mediated inhibition of WT1 expression reduces PCa cell proliferation and blocks cell cycle progression. ( a ) Relative cell viability of 22Rv and LNCaP PCa cells measured via cell titer assay at 3 d post-transfection with WT1 siRNA or si-NC (20 nM). Validation of WT1 knockdown see Fig. B. Error bars = SD; n = 3; ** p < 0.005. ( b ) Proliferation of 22Rv1 and LNCaP PCa cells (cell index) measured using the xCELLigence system post WT1 siRNA or si-NC transfection (20 nM). Validation of WT1 knockdown see Fig. C. Error bars = SD; n = 3. ( c ) Colony formation assay of 22Rv1 and LNCaP PCa cells 14–21 days post WT1 siRNA or si-NC (20 nM) transfection. Validation of WT1 knockdown see Fig. C. Error bars = SD; n = 3; ** p < 0.005. ( d ) Flow cytometry cell cycle analysis of 22Rv1 and LNCaP PCa cells transfected with WT1 siRNA or si-NC (20 nM) for 72 h. Validation of WT1 knockdown see Fig. D. n = 3; * p < 0.05, ** p < 0.005 relative to si-NC. ( e ) Western blot analysis of p21 (22Rv1 and LNCaP) and p53 (22Rv1) protein expression 72 h post-transfection of PCa cells with 20 nM WT1 siRNA or si-NC. β-actin is the loading control. Validation of WT1 knockdown see Fig. E. For full-length, non-cropped blots see Fig. B and S1C. n = 3. ( f ) Relative cell viability of 22Rv1 and LNCaP PCa cells measured via cell titer assay at 5 days post-transfection with WT1 siRNA or si-NC (20 nM), and 3 d post-17-AAG treatment (1 µM). Validation of WT1 knockdown see Fig. B. Error bars = SD; n = 3; ** p < 0.005.

Article Snippet: Human WT1 cDNA (WT1-203, ENST00000379079.8, Ensembl) was synthesized (GenScript) and cloned into LeGO-iT2 lentiviral vector (a gift from Boris Fehse, Addgene plasmid # 27343).

Techniques: Inhibition, Expressing, Titer Assay, Transfection, Biomarker Discovery, Knockdown, Colony Assay, Flow Cytometry, Cell Cycle Assay, Western Blot, Control

Journal: Scientific Reports

Article Title: The tumor suppressor miR-642a-5p targets Wilms Tumor 1 gene and cell-cycle progression in prostate cancer

doi: 10.1038/s41598-021-97190-x

Figure Lengend Snippet: WT1 overexpression increases colony formation and miR-642a-5p rescues this effect. ( a ) RT-qPCR analysis of WT1 gene expression following stable or transient LeGO-iT2-WT1-203 or LeGO-iT2-Empty plasmids, and overexpression of miR-642a-5p or miR-NC in 22Rv1 and LNCaP PCa cells. Expression of WT1 is normalized to HPRT housekeeping gene expression, calculated using the 2 −ΔΔCt method, and relative to Empty vector + miR-NC. Error bars = SE; n = 3; * p < 0.05, ** p < 0.005 relative to Empty vector + miR-NC. # p < 0.05 WT1-203 + miR-NC relative to WT1-203 + miR-642a-5p. ( b ) Colony formation assay of 22Rv1 PCa cells 14 days post transient WT1 overexpression/empty vector transfection and miR-NC/miR-642a-5p (30 nM) co-transfection. Error bars = SD; n = 3; ** p < 0.005 relative to Empty vector + miR-NC. ## p < 0.005 WT1-203 + miR-NC relative to WT1-203 + miR-642a-5p.

Article Snippet: Human WT1 cDNA (WT1-203, ENST00000379079.8, Ensembl) was synthesized (GenScript) and cloned into LeGO-iT2 lentiviral vector (a gift from Boris Fehse, Addgene plasmid # 27343).

Techniques: Over Expression, Quantitative RT-PCR, Gene Expression, Expressing, Plasmid Preparation, Colony Assay, Transfection, Cotransfection