plasmid pcdna3  (Addgene inc)

Journal: Experimental & Molecular Medicine

Article Title: AZIN1 RNA editing alters protein interactions, leading to nuclear translocation and worse outcomes in prostate cancer

doi: 10.1038/s12276-022-00845-6

Figure Lengend Snippet: The importance of the unedAZIN1 control was established by overexpressing wtAZIN1 or unedAZIN1 (2.5 µg plasmid for 24 h) in wtHEK293T and ADAR1 KO HEK293T cells. The samples were analyzed ( a ) by western blotting with the indicated antibodies and ( b ) with an MTT assay. The effect of overexpressing various AZIN1 alleles on the behavior of human prostate cancer PC3 or DU145 cells with regard to ( c and d ) invasion into the extracellular matrix, e and f cell proliferation, and ( g and h ) colony formation in soft agar. a – h PC3 and DU145 cells (as indicated in the figure) were transfected with plasmids expressing pCDNA3.1 (Empty); wild-type AZIN1 (wtAZIN1), which can be edited endogenously; pseudoedited AZIN1 (edAZIN); or AZIN1 with an uneditable codon 367 (uneditable AZIN1). Proliferation was measured by MTT reduction. Invasion through Matrigel supported by a Transwell membrane (8 μm pores) was assessed after methanol fixation and toluene blue staining. Soft agar colony formation was assessed by incubation for 19 days, fixation, and crystal violet staining. In ( b – h ), the bars indicate the mean (±S.D.) from three independent experiments. * significant difference compared to empty vector ( P < 0.05), (or compared to wtAZIN1 as in ( b )), ** significant difference compared to empty vector ( P < 0.01), *** significant difference compared to empty vector ( P < 0.001).

Article Snippet: The other plasmids used, pcDNA3.1-Clover-mRuby2 (plasmid #49089) and pcDNA3 mRuby2 LIC cloning vector (6H), were commercially available (Addgene, MA, USA).

Techniques: Control, Plasmid Preparation, Western Blot, MTT Assay, Transfection, Expressing, Membrane, Staining, Incubation

Journal: Experimental & Molecular Medicine

Article Title: AZIN1 RNA editing alters protein interactions, leading to nuclear translocation and worse outcomes in prostate cancer

doi: 10.1038/s12276-022-00845-6

Figure Lengend Snippet: a , PC3 and ( b ) human embryonic kidney (HEK293) cells were transfected with 1.5–2.5 µg of pcDNA3.1-Clover-AZIN1, pcDNA3.1-Clover-edited-AZIN1 or Clover-uneditable-AZIN1 for 24 h and analyzed by confocal microscopy. c The amount of edAZIN1 was measured by ddPCR, and tissues with low and high AZIN1 editing were identified (tissues with low editing had < 1% edAZIN1 among total AZIN1 RNA, while tissues with high editing had up to 31.5%). Then, the same tissues were stained with an anti-AZIN1 antibody ( d ). Tissues with low AZIN-1 editing showed cytoplasmic localization only (up), while tissues with high AZIN1 editing showed cytoplasmic and nuclear localization of AZIN1 (down). The intensity was measured by ImageJ software, and the anti-AZIN1 antibody was validated as described in the Materials and Methods section. In ( a and b ), the bar graphs summarize the percentages of 50 cells exhibiting intense nuclear localization of antizyme + /− 95% confidence intervals. Measurements were performed in a blinded manner. In each case, nuclear localization was significantly associated with transfection with the edAZIN plasmid ( P < 0.0001 by Fisher’s exact test).

Article Snippet: The other plasmids used, pcDNA3.1-Clover-mRuby2 (plasmid #49089) and pcDNA3 mRuby2 LIC cloning vector (6H), were commercially available (Addgene, MA, USA).

Techniques: Transfection, Confocal Microscopy, Staining, Software, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: AZIN1 RNA editing alters protein interactions, leading to nuclear translocation and worse outcomes in prostate cancer

doi: 10.1038/s12276-022-00845-6

Figure Lengend Snippet: Using CRISPR/Cas9 gene editing and specific sgRNAs, Myosin 9 protein expression was successfully knocked out in PC3 ( a ) and DU145 ( c ) cells. Myosin 9-knockout PC3 ( b ) and DU145 ( d ) cells were transfected with 2,5 µg of pcDNA3.1-Clover-edited-AZIN1 for 24 h and analyzed by confocal microscopy. The effect of overexpressing the edAZIN1 allele in Myosin 9 knockout PC3 and DU145 cells with regard to cell proliferation ( e and f ), invasion into the extracellular matrix ( g and h ), and colony formation in soft agar ( i ). Proliferation was measured by MTT reduction. Invasion through Matrigel supported by a Transwell membrane (8 μm pores) was assessed after methanol fixation and toluene blue staining. Soft agar colony formation was assessed by incubation for 19–21 days, fixation, and crystal violet staining. The morphology of PC3 ( j ) and DU14 ( k ) cells is shown, and representative images acquired by light microscopy (lens: original magnification, 10X) are presented. The bars indicate the mean (±S.D.) from three independent experiments. * significant difference compared to empty vector ( P < 0.05), ** significant difference compared to empty vector ( P < 0.01) and *** significant difference compared to empty vector ( P < 0.001). Myosin 9 knockout PC3 ( l ) and DU145 ( m ) cell populations were analyzed by western blotting with the indicated antibodies.

Article Snippet: The other plasmids used, pcDNA3.1-Clover-mRuby2 (plasmid #49089) and pcDNA3 mRuby2 LIC cloning vector (6H), were commercially available (Addgene, MA, USA).

Techniques: CRISPR, Expressing, Knock-Out, Transfection, Confocal Microscopy, Membrane, Staining, Incubation, Light Microscopy, Plasmid Preparation, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: AZIN1 RNA editing alters protein interactions, leading to nuclear translocation and worse outcomes in prostate cancer

doi: 10.1038/s12276-022-00845-6

Figure Lengend Snippet: a The binding affinities of AZ for 3 AZIN1 position 367 point mutants were measured by a FRET assay with titration of AZ-mRuby2 (100 pM-1 µM) to a fixed concentration of Clover-AZIN1 variants (50 nM). b The data were fitted using nonlinear regression to a four-parameter binding isotherm to determine the equilibrium dissociation constant (Kd) of the protein‒protein interaction.

Article Snippet: The other plasmids used, pcDNA3.1-Clover-mRuby2 (plasmid #49089) and pcDNA3 mRuby2 LIC cloning vector (6H), were commercially available (Addgene, MA, USA).

Techniques: Binding Assay, Titration, Concentration Assay