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Addgene inc pcdna3 1 clover mruby2
Experimental setup and gating strategy of FRET measurements. Clover, <t>mRuby2,</t> Clover + mRuby2 and Clover fused (63bp) mRuby2 protein expression are shown in total HEK293T cell lysates by Western blot analysis against GFP (A) and tRFP (B) . Fluorescence microscopy images (C) of Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 expressing HEK293T cells. Panel 1 (1) shows the brightfield. Cells expressing mRuby2 are depicted in panel 2 (2), Clover-positive cells in panel 3 (3). Cell nuclei were counterstained with Hoechst 33342 (panel 4 (4)). An overlay to estimate cytosolic and nuclear region is provided in panel 5 (5). FLIM images (D) of HEK293T cells with the controls Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2. The histogram (E) shows the normalized frequency of fluorescence lifetimes in the images. Experimental setup and gating strategy to measure FRET by flow cytometry are depicted in living cells (F) . HEK293T cells were stably transduced with the controls Clover, mRuby2, Clover + mRuby2 as well as the Clover fused (63bp) mRuby2 and analyzed using a flow cytometer. Double positive cells were gated in panel 1 (1). False-positive FRET signals resulting from mRuby2 excitation by the 488 nm laser were excluded in panel 2 (2). In panel 3 (3), the remaining cells were evaluated for FRET by adjusting a gate defining cells which were co-transduced with Clover + mRuby2 and should be FRET-negative. The numbers in panel 3 (3) give total percentages of FRET-positive HEK293T cells. Images and flow cytometry-plots are representative for experiments which were performed at least three times. (G) FRET efficiencies were determined as described in Materials and Methods by analyzing Clover/mRuby2-double positive cells only. Experiments were performed at least three times. *** P ≤ 0.001.
Pcdna3 1 Clover Mruby2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc pcdna3 mruby2 lic cloning vector 6h
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 <t>pCDNA3.1</t> (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).
Pcdna3 Mruby2 Lic Cloning Vector 6h, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc pcdna3 1 clovermruby2
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 <t>pCDNA3.1</t> (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).
Pcdna3 1 Clovermruby2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc k beam
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 <t>pCDNA3.1</t> (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).
K Beam, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Addgene inc kurt beam addgene plasmid 49089
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 <t>pCDNA3.1</t> (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).
Kurt Beam Addgene Plasmid 49089, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Experimental setup and gating strategy of FRET measurements. Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 protein expression are shown in total HEK293T cell lysates by Western blot analysis against GFP (A) and tRFP (B) . Fluorescence microscopy images (C) of Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 expressing HEK293T cells. Panel 1 (1) shows the brightfield. Cells expressing mRuby2 are depicted in panel 2 (2), Clover-positive cells in panel 3 (3). Cell nuclei were counterstained with Hoechst 33342 (panel 4 (4)). An overlay to estimate cytosolic and nuclear region is provided in panel 5 (5). FLIM images (D) of HEK293T cells with the controls Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2. The histogram (E) shows the normalized frequency of fluorescence lifetimes in the images. Experimental setup and gating strategy to measure FRET by flow cytometry are depicted in living cells (F) . HEK293T cells were stably transduced with the controls Clover, mRuby2, Clover + mRuby2 as well as the Clover fused (63bp) mRuby2 and analyzed using a flow cytometer. Double positive cells were gated in panel 1 (1). False-positive FRET signals resulting from mRuby2 excitation by the 488 nm laser were excluded in panel 2 (2). In panel 3 (3), the remaining cells were evaluated for FRET by adjusting a gate defining cells which were co-transduced with Clover + mRuby2 and should be FRET-negative. The numbers in panel 3 (3) give total percentages of FRET-positive HEK293T cells. Images and flow cytometry-plots are representative for experiments which were performed at least three times. (G) FRET efficiencies were determined as described in Materials and Methods by analyzing Clover/mRuby2-double positive cells only. Experiments were performed at least three times. *** P ≤ 0.001.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Experimental setup and gating strategy of FRET measurements. Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 protein expression are shown in total HEK293T cell lysates by Western blot analysis against GFP (A) and tRFP (B) . Fluorescence microscopy images (C) of Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2 expressing HEK293T cells. Panel 1 (1) shows the brightfield. Cells expressing mRuby2 are depicted in panel 2 (2), Clover-positive cells in panel 3 (3). Cell nuclei were counterstained with Hoechst 33342 (panel 4 (4)). An overlay to estimate cytosolic and nuclear region is provided in panel 5 (5). FLIM images (D) of HEK293T cells with the controls Clover, mRuby2, Clover + mRuby2 and Clover fused (63bp) mRuby2. The histogram (E) shows the normalized frequency of fluorescence lifetimes in the images. Experimental setup and gating strategy to measure FRET by flow cytometry are depicted in living cells (F) . HEK293T cells were stably transduced with the controls Clover, mRuby2, Clover + mRuby2 as well as the Clover fused (63bp) mRuby2 and analyzed using a flow cytometer. Double positive cells were gated in panel 1 (1). False-positive FRET signals resulting from mRuby2 excitation by the 488 nm laser were excluded in panel 2 (2). In panel 3 (3), the remaining cells were evaluated for FRET by adjusting a gate defining cells which were co-transduced with Clover + mRuby2 and should be FRET-negative. The numbers in panel 3 (3) give total percentages of FRET-positive HEK293T cells. Images and flow cytometry-plots are representative for experiments which were performed at least three times. (G) FRET efficiencies were determined as described in Materials and Methods by analyzing Clover/mRuby2-double positive cells only. Experiments were performed at least three times. *** P ≤ 0.001.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Expressing, Western Blot, Fluorescence, Microscopy, Flow Cytometry, Stable Transfection, Transduction

Determination of Clover-PPARγ1, mRuby2-RXRα and mRuby2-RXRα Δ414-462 protein expression. Graphical scheme of the structure of the full length human PPARγ1 protein (A) ; the full length human RXRα protein (B) and the human RXRα deletion construct with a C-terminal absence of the sequence AA 414-462 (C) . PPARγ1 was N-terminally labeled with the green fluorophore Clover and both of the two RXRα constructs N-terminally with the red fluorophore mRuby2. Clover-PPARγ1, mRuby2-RXRα and mRuby2-RXRα Δ414-462 protein expressions are shown in total HEK293T cell lysates visualized by Western blot analysis against human PPARγ (D) , GFP (E) , human RXRα (F) and tRFP (G) . Images are representative of experiments which were performed at least three times.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Determination of Clover-PPARγ1, mRuby2-RXRα and mRuby2-RXRα Δ414-462 protein expression. Graphical scheme of the structure of the full length human PPARγ1 protein (A) ; the full length human RXRα protein (B) and the human RXRα deletion construct with a C-terminal absence of the sequence AA 414-462 (C) . PPARγ1 was N-terminally labeled with the green fluorophore Clover and both of the two RXRα constructs N-terminally with the red fluorophore mRuby2. Clover-PPARγ1, mRuby2-RXRα and mRuby2-RXRα Δ414-462 protein expressions are shown in total HEK293T cell lysates visualized by Western blot analysis against human PPARγ (D) , GFP (E) , human RXRα (F) and tRFP (G) . Images are representative of experiments which were performed at least three times.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Expressing, Construct, Sequencing, Labeling, Western Blot

Analysis of protein-protein interactions between Clover-PPARγ1, mRuby2-RXRα and mRuby2-RXRα Δ414-462. A PPARγ‑dependent transactivation assay was used in HEK293T cells to verify the mechanistic functionality of the Clover, Clover-PPARγ1, mRuby2, mRuby2-RXRα and mRuby2-RXRα Δ414-462 (A and B) . HEK293T cells expressing protein(s) as indicated were stimulated for 24 h with 1 µM rosiglitazone and 10 µM GW9662 alone or in combination. Values from PPARγ‑dependent transactivation experiments are means ± SD of three to ten individual experiments. Each PPARγ‑dependent transactivation assay experiment was performed in quadruple. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. Fluorescence microscopy images of HEK293T cells expressing Clover-PPARγ1, mRuby2-RXRα, mRuby2-RXRα Δ414-462, Clover-PPARγ1 + mRuby2-RXRα and Clover-PPARγ1 + mRuby2-RXRα Δ414-462 are depicted (C) . The brightfield is shown in panel 1 (1). The mRuby2-positive cells are depicted in panel 2 (2), Clover in panel 3 (3), Hoechst 33342-stained cell nuclei in panel 4 (4) and an overlay to estimate the localization is provided in panel 5 (5). Images are representative for experiments which were performed at least three times. The PCC was used for the correlation quantification of the subcellular co-localization of Clover-PPARγ1 expressed in combination with mRuby2-RXRα or mRuby2-RXRα Δ414-462 (D) . R-Values are means ± SD of at least 18 individual cells. *** P ≤ 0.001. Representative primary flow cytometry-plots from an experiment which was performed three times showing the amount of FRET-positive living HEK293T cells expressing Clover-PPARγ1 + mRuby2-RXRα and Clover-PPARγ1 + mRuby2-RXRα Δ414-462 (E) . The numbers in panel 3 (3) give total percentages of HEK293T cells within the FRET gate. A comparison of the total percentages of FRET-positive living HEK293T Clover-PPARγ1 + mRuby2-RXRα and HEK293T Clover-PPARγ1 + mRuby2-RXRα Δ414-462 cells is depicted in (F). Flow cytometry-based FRET efficiencies, depicted in (G) , are calculated as described in Materials and Methods. Values are means ± SD of three experiments. ** P ≤ 0.01, *** P ≤ 0.001.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Analysis of protein-protein interactions between Clover-PPARγ1, mRuby2-RXRα and mRuby2-RXRα Δ414-462. A PPARγ‑dependent transactivation assay was used in HEK293T cells to verify the mechanistic functionality of the Clover, Clover-PPARγ1, mRuby2, mRuby2-RXRα and mRuby2-RXRα Δ414-462 (A and B) . HEK293T cells expressing protein(s) as indicated were stimulated for 24 h with 1 µM rosiglitazone and 10 µM GW9662 alone or in combination. Values from PPARγ‑dependent transactivation experiments are means ± SD of three to ten individual experiments. Each PPARγ‑dependent transactivation assay experiment was performed in quadruple. * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001. Fluorescence microscopy images of HEK293T cells expressing Clover-PPARγ1, mRuby2-RXRα, mRuby2-RXRα Δ414-462, Clover-PPARγ1 + mRuby2-RXRα and Clover-PPARγ1 + mRuby2-RXRα Δ414-462 are depicted (C) . The brightfield is shown in panel 1 (1). The mRuby2-positive cells are depicted in panel 2 (2), Clover in panel 3 (3), Hoechst 33342-stained cell nuclei in panel 4 (4) and an overlay to estimate the localization is provided in panel 5 (5). Images are representative for experiments which were performed at least three times. The PCC was used for the correlation quantification of the subcellular co-localization of Clover-PPARγ1 expressed in combination with mRuby2-RXRα or mRuby2-RXRα Δ414-462 (D) . R-Values are means ± SD of at least 18 individual cells. *** P ≤ 0.001. Representative primary flow cytometry-plots from an experiment which was performed three times showing the amount of FRET-positive living HEK293T cells expressing Clover-PPARγ1 + mRuby2-RXRα and Clover-PPARγ1 + mRuby2-RXRα Δ414-462 (E) . The numbers in panel 3 (3) give total percentages of HEK293T cells within the FRET gate. A comparison of the total percentages of FRET-positive living HEK293T Clover-PPARγ1 + mRuby2-RXRα and HEK293T Clover-PPARγ1 + mRuby2-RXRα Δ414-462 cells is depicted in (F). Flow cytometry-based FRET efficiencies, depicted in (G) , are calculated as described in Materials and Methods. Values are means ± SD of three experiments. ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Transactivation Assay, Expressing, Fluorescence, Microscopy, Staining, Flow Cytometry

Protein expression analysis of Clover-PPARγ1 and N-CoR2-mRuby2 constructs. Graphical scheme of the structure of the full-length human N‑CoR2 protein (A) and the used N-CoR2 constructs, N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2 (amplified out of HEK293T cells cDNA), N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 (B) . All N-CoR2 constructs were C-terminally labeled with the red fluorophore mRuby2. Western blot analysis of total lysate of HEK293T cells stably expressing Clover-PPARγ1, N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 alone or in combination against human PPARγ1 ( C ), GFP (D) , human N-CoR2 ( E ) and tRFP (F) . Images are representative of experiments which were performed at least three times.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Protein expression analysis of Clover-PPARγ1 and N-CoR2-mRuby2 constructs. Graphical scheme of the structure of the full-length human N‑CoR2 protein (A) and the used N-CoR2 constructs, N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2 (amplified out of HEK293T cells cDNA), N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 (B) . All N-CoR2 constructs were C-terminally labeled with the red fluorophore mRuby2. Western blot analysis of total lysate of HEK293T cells stably expressing Clover-PPARγ1, N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 alone or in combination against human PPARγ1 ( C ), GFP (D) , human N-CoR2 ( E ) and tRFP (F) . Images are representative of experiments which were performed at least three times.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Expressing, Construct, Amplification, Labeling, Western Blot, Stable Transfection

Mechanistic functionality and cellular localization of Clover-PPARγ1 and N-CoR2-mRuby2 constructs. A PPARγ‑dependent transactivation assay was used to verify the mechanistic functionality of HEK293T cells expressing Clover or Clover-PPARγ1 in combination with N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 (A) . HEK293T cells expressing protein(s) as indicated were stimulated for 24 h with 1 µM rosiglitazone. Values from PPARγ‑dependent transactivation experiments are means ± SD of four to 12 individually experiments. Each PPARγ‑dependent transactivation assay experiment was performed in quadruple. * P ≤ 0.05. Fluorescence microscopy images of HEK293T cells expressing N-CoR2 WT-mRuby2 and Clover-PPARγ1 in combination with N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 cells are depicted (B) . The brightfield is shown in panel 1 (1). The mRuby2-positive cells are depicted in panel 2 (2), Clover in panel 3 (3), Hoechst 33342-stained cell nuclei in panel 4 (4) and an overlay to estimate the localization is provided in panel 5 (5). Images are representative of experiments which were performed at least three times. The PCC was used for the correlation quantification of the sub-cellular co-localization of Clover-PPARγ1 expressed in combination with N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 in HEK293T cells (C) . R-Values are means ± SD of at least seven individual cells. *** P ≤ 0.001.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Mechanistic functionality and cellular localization of Clover-PPARγ1 and N-CoR2-mRuby2 constructs. A PPARγ‑dependent transactivation assay was used to verify the mechanistic functionality of HEK293T cells expressing Clover or Clover-PPARγ1 in combination with N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 (A) . HEK293T cells expressing protein(s) as indicated were stimulated for 24 h with 1 µM rosiglitazone. Values from PPARγ‑dependent transactivation experiments are means ± SD of four to 12 individually experiments. Each PPARγ‑dependent transactivation assay experiment was performed in quadruple. * P ≤ 0.05. Fluorescence microscopy images of HEK293T cells expressing N-CoR2 WT-mRuby2 and Clover-PPARγ1 in combination with N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 cells are depicted (B) . The brightfield is shown in panel 1 (1). The mRuby2-positive cells are depicted in panel 2 (2), Clover in panel 3 (3), Hoechst 33342-stained cell nuclei in panel 4 (4) and an overlay to estimate the localization is provided in panel 5 (5). Images are representative of experiments which were performed at least three times. The PCC was used for the correlation quantification of the sub-cellular co-localization of Clover-PPARγ1 expressed in combination with N-CoR2 WT-mRuby2, N-CoR2 (ΔID1 exon)-mRuby2, N-CoR2 (ΔID1 CoRNR box)-mRuby2 and N-CoR2 (ΔID2 CoRNR box)-mRuby2 in HEK293T cells (C) . R-Values are means ± SD of at least seven individual cells. *** P ≤ 0.001.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Construct, Transactivation Assay, Expressing, Fluorescence, Microscopy, Staining

Flow cytometry-based FRET-measurements to analyze protein-protein interactions between Clover-PPARγ1 and N-CoR2-mRuby2 constructs. Representative primary flow cytometry-plots showing the quantity of FRET-positive living HEK293T Clover-PPARγ1 + N-CoR2 WT-mRuby2 cells are depicted (A). Images are representative of experiments which were performed three times. The cells were cultured for 24 h with DMSO, 1 µM of the PPARγ agonist, rosiglitazone, and 10 µM of the PPARγ antagonist, GW9662, alone or in combination. Panel 1 (1) represents the gating for Clover/mRuby2-double positive cells. Panel 2 (2) depicts cells positive for the first FRET gate of FRET 488/610nm vs. mRuby2 561/610 nm and panel 3 (3) shows cells with a FRET signal. Numbers in panels (2) and (3) represent total percentages of cells within the FRET gates. A comparison of the total percentages of FRET-positive living HEK293T Clover-PPARγ1 + N-CoR2 WT-mRuby2 cells is depicted in (B). The relative flow cytometry-based FRET efficiencies calculated as described in Materials and Methods are presented in (C). Comparison of the total percentages of FRET-positive living HEK293T Clover-PPARγ1 + N-CoR2 WT-mRuby2, Clover-PPARγ1 + N-CoR2 (ΔID1 exon)-mRuby2, Clover-PPARγ1 + N-CoR2 (ΔID1 CoRNR box)-mRuby2 and Clover-PPARγ1 + N-CoR2 (ΔID2 CoRNR box)-mRuby2 cells are depicted in (D). Flow cytometry-based efficiencies of Clover/mRuby2-double positive cells, calculated as described in Materials and Methods, are shown in (E). Values are means ± SD of three experiments. ** P ≤ 0.01 and *** P ≤ 0.001.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Flow cytometry-based FRET-measurements to analyze protein-protein interactions between Clover-PPARγ1 and N-CoR2-mRuby2 constructs. Representative primary flow cytometry-plots showing the quantity of FRET-positive living HEK293T Clover-PPARγ1 + N-CoR2 WT-mRuby2 cells are depicted (A). Images are representative of experiments which were performed three times. The cells were cultured for 24 h with DMSO, 1 µM of the PPARγ agonist, rosiglitazone, and 10 µM of the PPARγ antagonist, GW9662, alone or in combination. Panel 1 (1) represents the gating for Clover/mRuby2-double positive cells. Panel 2 (2) depicts cells positive for the first FRET gate of FRET 488/610nm vs. mRuby2 561/610 nm and panel 3 (3) shows cells with a FRET signal. Numbers in panels (2) and (3) represent total percentages of cells within the FRET gates. A comparison of the total percentages of FRET-positive living HEK293T Clover-PPARγ1 + N-CoR2 WT-mRuby2 cells is depicted in (B). The relative flow cytometry-based FRET efficiencies calculated as described in Materials and Methods are presented in (C). Comparison of the total percentages of FRET-positive living HEK293T Clover-PPARγ1 + N-CoR2 WT-mRuby2, Clover-PPARγ1 + N-CoR2 (ΔID1 exon)-mRuby2, Clover-PPARγ1 + N-CoR2 (ΔID1 CoRNR box)-mRuby2 and Clover-PPARγ1 + N-CoR2 (ΔID2 CoRNR box)-mRuby2 cells are depicted in (D). Flow cytometry-based efficiencies of Clover/mRuby2-double positive cells, calculated as described in Materials and Methods, are shown in (E). Values are means ± SD of three experiments. ** P ≤ 0.01 and *** P ≤ 0.001.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Flow Cytometry, Construct, Cell Culture

Transfer of the flow cytometry-based FRET system to J774A.1 macrophages. Flow cytometry-based FRET efficiency was determined in murine J774A.1 macrophages following transduction with (A) Clover + mRuby2 or Clover fused (63bp) mRuby2 and (B) Clover-PPARγ1 in combination with N-CoR2 WT-mRuby2 or N-CoR2 (ΔID exon)-mRuby2. Flow cytometry-based FRET efficiencies of Clover/mRuby2-double positive cells were calculated as described in Materials and Methods.

Journal: Theranostics

Article Title: Flow cytometry-based FRET identifies binding intensities in PPARγ1 protein-protein interactions in living cells

doi: 10.7150/thno.29367

Figure Lengend Snippet: Transfer of the flow cytometry-based FRET system to J774A.1 macrophages. Flow cytometry-based FRET efficiency was determined in murine J774A.1 macrophages following transduction with (A) Clover + mRuby2 or Clover fused (63bp) mRuby2 and (B) Clover-PPARγ1 in combination with N-CoR2 WT-mRuby2 or N-CoR2 (ΔID exon)-mRuby2. Flow cytometry-based FRET efficiencies of Clover/mRuby2-double positive cells were calculated as described in Materials and Methods.

Article Snippet: Therefore, the coding regions of Clover, mRuby2 and by a 63bp-linker fused Clover-mRuby were amplified by polymerase chain reaction (PCR) from pcDNA3-Clover (Addgene#40259), pcDNA3-mRuby2 (Addgene#40260) and pcDNA3.1-Clover-mRuby2 (Addgene#49089) elongated with the additional bases of 5´-CGCCCGGGGGGGATCGCCGCCACC-3´ on their 5´ end and 5´-CCTGCAGGCATGCAA-3´ on their 3´ end for subsequent recombination into the BamHI-SbfI linearized target vector by In-Fusion ® HD cloning (Clontech Laboratories, Inc., Mountain View, USA).

Techniques: Flow Cytometry, Transduction

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).

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

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).

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

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.

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, Staining, Incubation, Light Microscopy, Plasmid Preparation, Western Blot

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.

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