Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: L1-70 interacts with TOP1, PPARγ and NDUFV2, but not with other L1 binding partners in neurons. Cultured cortical neurons were treated with vehicle (-) or with the serine protease inhibitor aprotinin. They were then subjected to proximity ligation with L1 antibody and antibodies against SFPQ, NonO, PSPC1, WDR5, TOP1, HistH1, Nup93, Hsc70, SYT1, impβ1, ERα, RXR, PPARγ, NDUFV2, AR, VDR or GAPDH. ( a ) Representative images of vehicle- and aprotinin-treated neurons stained with the mouse L1 antibody C-2 and rabbit antibodies against TOP1, PPARγ or NDUFV2 are shown. Nuclei are stained with DAPI. Scale bars: 10 µm. ( b ) Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of vehicle control set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( c ) Cortical neurons were treated with or without L1 antibody 557. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatment without antibody 557 set to 100%). (*** p < 0.005; one-way ANOVA with Bonferroni’s multiple comparison test). ( d ) Cortical and cerebellar neurons pretreated with vehicle or aprotinin were incubated without or with antibody 557 in the absence or presence of aprotinin. Mean values + SD from two independent experiments are shown for the average numbers of red dots per cell relative to control (values of treatments without aprotinin set to 100%) (**** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Binding Assay, Cell Culture, Protease Inhibitor, Ligation, Staining, Control, Comparison, Incubation

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: L1-ICD binds to TOP1 and PPARγ. Recombinant TOP1 ( a ) and PPARγ ( b ) were substrate-coated and incubated with increasing concentrations of L1-ICD. Binding of L1-ICD was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values ± SD from three independent experiments carried out in triplicate are shown. ( c ) Recombinant TOP1 and PPARγ were substrate-coated and incubated with L1-ICD in the absence or presence of a five-fold excess of L1 peptides P1, P2, P3, P4 or P5, which cover the entire L1-ICD sequence. The ICD and transmembrane domain (TMD) of L1 and the positions of the peptides in L1-ICD are visualized. Binding was determined by ELISA using mouse L1 antibody C-2 and horseradish peroxidase-conjugated secondary antibody. Mean values + SD from five independent experiments carried out in triplicate are shown for the binding relative to control (values in the absence of peptides set to 100%) (** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test).

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: No enhanced interaction of L1 with TOP1, PPARγ and NDUFV2 in stimulated cortical neurons from L1-70-lacking mutant mice. Cultured cortical neurons from L1/687 ( a ) and L1/858–863 ( b ) mutant mice and their wild-type littermates ( a , b ) were treated without (-) or with L1 antibody 557 (557) and then subjected to proximity ligation assay with L1 antibody and TOP1, PPARγ or NDUFV2 antibodies. ( a ) For control, the L1 antibody and an antibody against SFPQ, ERα or RXR were used for proximity ligation. ( a , b ) Mean values + SD are shown for the average numbers of L1/TOP1-, L1/NDUFV2-, L1/PPARγ-, L1/SFPQ-, L1/ERα-, and L1/RXR-positive dots per cell relative to control (values of unstimulated wild-type neurons set to 100%) (** p < 0.01, **** p < 0.001 relative to non-stimulated wild-type neurons, ## p < 0.01, #### p < 0.001 relative to stimulated wild-type neurons; one-way ANOVA with Dunn’s multiple comparison test). ( c ) Non-nuclear brain fractions from wild-type (WT) and L1/858–863 mutant (Mut) mice were subjected to immunoprecipitation (IP) with immobilized mouse antibodies against TOP1, PPARγ and NDUFV2. Non-nuclear fractions (input) and immunoprecipitates were subjected to Western blot (WB) analysis with L1 antibody C-2. The arrowhead indicates L1-70.

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Mutagenesis, Cell Culture, Proximity Ligation Assay, Control, Ligation, Comparison, Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: Reduction of TOP1, PPARγ or NDUFV2 expression inhibits L1-dependent neurite outgrowth. ( a , b ) Cortical neurons were mock-transfected or transfected with control siRNA, TOP1 siRNA, PPARγ siRNA or NDUFV2 siRNA. Neurons were then treated without or with L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, ### p < 0.005, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test).

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: The interaction of L1 with TOP1, PPARγ or NDUFV2 is reduced by L1, TOP1, PPARγ and NDUFV2 siRNAs. ( a ) Cultured cortical neurons were mock-transfected or transfected with TOP1, PPARγ, NDUFV2 or L1 siRNAs. The transfected cells were then subjected to proximity ligation with L1 antibody and antibodies against TOP1, PPARγ or NDUFV2. Mean values + SD from two experiments are shown for the average numbers of L1/TOP1-, L1/PPARγ- and L1/NDUFV2-positive red dots per cell relative to control (values of mock-transfected neurons set to 100%) (* p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001; one-way ANOVA with Bonferroni’s multiple comparison test). ( b ) Western blot analysis of lysates from mock-transfected neurons or neurons transfected with TOP1 siRNA using TOP1 antibody. Ponceau S staining of a prominent 35 kDa band served as loading control. ( c ) Mock-transfected neurons or neurons transfected with PPARγ or NDUFV2 siRNAs were subjected to immunostaining with PPARγ or NDUFV2 antibody. Nuclei are stained with DAPI. Representative images are shown and indicate reduced PPARγ or NDUFV2 levels in neurons transfected with PPARγ siRNA or NDUFV2 siRNA. Scale bars: 10 µm.

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, Transfection, Ligation, Control, Comparison, Western Blot, Staining, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: Disturbance of the L1/TOP1 and L1/PPARγ interactions inhibits L1-dependent neurite outgrowth from cortical neurons. ( a , b ) Cortical neurons were treated without (-) or with tat-P4 peptide. Neurons were then treated without or with L1 antibody 557 (557). ( a ) Mean values + SEM from three independent experiments are shown for total neurite lengths (**** p < 0.001 relative to mock-transfected non-stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Dunn’s multiple comparison test). ( b ) After the treatments, neurons were subjected to proximity ligation with L1 antibody and TOP1 antibody or PPARγ antibody. Mean values + SD from three independent experiments are shown for average numbers of L1/TOP1- and L1/PPARγ-positive red dots per cell relative to control (values of non-transfected neurons set to 100%) (*** p < 0.005, **** p < 0.001 relative to mock-transfected stimulated neurons, #### p < 0.001 relative to mock-transfected stimulated neurons; one-way ANOVA with Bonferroni’s multiple comparison test).

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Transfection, Comparison, Ligation, Control

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: Topoisomerase inhibitors reduce L1-dependent neurite outgrowth as well as neuronal survival and migration. Cerebellar neurons ( a , c ) and cerebellar explants ( e ), and cortical neurons ( b , d ) were treated without (-) or with topotecan (Topo) or irinotecan (Irino) in the absence or presence of L1 antibody 557. ( a , b ) Mean values + SEM from three independent experiments are shown for total neurite lengths (*** p < 0.005, **** p < 0.001 relative to non-stimulated neurons in the absence of inhibitors, ### p < 0.005, #### p < 0.001 relative to stimulated neurons in the absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( c , d ) Mean values + SEM from three independent experiments are shown for the numbers of viable cells relative to control (values of unstimulated neurons in the absence of H 2 O 2 set to 100%) (#### p < 0.0001 relative to stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , **** p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and the presence of H 2 O 2 , §§§§ p < 0.0001 relative to non-stimulated neurons in absence of inhibitors and H 2 O 2 ; one-way ANOVA with Dunn’s multiple comparison test). ( e ) Mean values + SEM from three independent experiments are shown for the total numbers of migrating cells (### p < 0.005 relative to stimulated neurons in absence of inhibitors, *** p < 0.005 relative to non-stimulated neurons in absence of inhibitors; one-way ANOVA with Dunn’s multiple comparison test). ( f ) Nuclear extracts from cortical neurons transfected without (mock) or with control siRNA, L1 siRNA or TOP1 siRNA were incubated with supercoiled pUC19 plasmid DNA. For control, the vector was incubated with the reaction buffer. The samples were then subjected to agarose gel electrophoresis and ethidium bromide staining. A representative image of a stained gel is shown.

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Migration, Comparison, Control, Transfection, Incubation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions

doi: 10.3390/ijms24032097

Figure Lengend Snippet: The L1/TOP1 interaction regulates the expression of the long autism genes Nrxn1 and Nlgn1 and the expression of the mitochondrial gene ND2. Cortical neurons were mock-transfected or transfected with L1 siRNA and subjected to RNA isolation and RT-qPCR 48 h after seeding. The mRNA levels of long autism ( a ) or mitochondrial ( b ) genes were normalized to the actin mRNA levels. ( c ) Cerebellar neurons from L1/858-863 mutant, L1/687 mutant and their wild-type littermates were maintained in culture for 48 h and subjected to RNA isolation and RT-qPCR. The ND2, Nrx1 and Nrx1 mRNA levels were normalized to the actin mRNA levels. Mean values + SD from five independent experiments with triplicates ( n = 5 mice) ( a , b ) or from six cultures per genotype ( n = 6 mice) ( c ) are shown for normalized mRNA levels in neurons after transfection with L1 siRNA relative to the levels in mock-transfected neurons ( a , b ) or in mutant neurons relative to wild-type neurons ( c ) (* p < 0.05, ** p < 0.01, *** p < 0.005; one-way ANOVA with Tukey’s ( a , b ) or Bonferroni’s ( c ) multiple comparison tests).

Article Snippet: Mouse L1 siRNA (NCAM-L1; sc-43173), mouse TOP1 siRNA (Topo I; sc-36693), mouse PPARγ siRNA (PPARgamma; sc-29456), mouse NDUFV2 siRNA (sc-149892), and control siRNA (Control siRNA-A; sc-37007) were from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR, Mutagenesis, Comparison

Journal: Science Advances

Article Title: RAD54L2 counters TOP2-DNA adducts to promote genome stability

doi: 10.1126/sciadv.adl2108

Figure Lengend Snippet: ( A ) Quantification of γH2AX foci in G 1 cells (cyclin A negative). Cells were treated with 30 μM etoposide for 30 min, washed, and left to recover for 4 or 8 hours. n > 3 independent experiments. Bars represent means ± SEM. One-way analysis of variance (ANOVA) was used for statistical testing. ( B ) DUST assays in CTRL or RAD54L2 KO HAP1 cells collected after 1 hour of treatment with 20 μM etoposide or 30 min or 1 hour after etoposide washout. This experiment was carried out three times with similar results. ( C ) Quantifications of TOP2α or TOP2β signal from the DUST assays. Bars represent means ± SEM. ( D ) Proposed mechanism for RAD54L2 in TOP2cc resolution. UBC9, SUMO conjugating enzyme 2 ligase. Figure generated with BioRender. ns, not significant.

Article Snippet: Antibodies used were as follows: RAD54L2 (Abcam, ab86063), TOP2α (Bethyl Laboratories, A300-054A), TOP2β (BD Biosciences, 611493), mCherry (Abcam, ab167453), tubulin (Sigma-Aldrich, T9026), TDP2 (Bethyl, A302-737A), ZNF451 (Sigma-Aldrich, SAB2108741), LIG4 (ab193353, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab8245, Abcam).

Techniques: Generated

Journal: International Journal of Molecular Sciences

Article Title: TNF-α-Induced YAP/TAZ Activity Mediates Leukocyte-Endothelial Adhesion by Regulating VCAM1 Expression in Endothelial Cells

doi: 10.3390/ijms19113428

Figure Lengend Snippet: YAP mediates TNF-α-induced VCAM1 and ICAM1 expression in endothelial cells. ( a ) HUVECs were transfected with 25 nM control scrambled (sc) or YAP/TAZ (Y+T) siRNA. After 48 h, cells were treated with TNF-α for 6 h. VCAM1 and ICAM1 protein levels were assessed by western blot analysis. ( b , c ) Relative expression of VCAM1 ( b ) and ICAM1 ( c ) protein after TNF-α treatment was quantified. ( d , e ) HUVECs were transfected with sc or Y+T siRNA. After 48 h, the cells were treated with TNF-α for 24 h. The relative levels of VCAM1 ( d ) and ICAM1 ( e ) mRNA were determined by qRT-PCR. ( f ) Cells were co-transfected with control, YAP and TAZ, or YAP S127A and TAZ S89A overexpressing vectors. After 24 h, the cells were treated with TNF-α for 6 h. VCAM1 and ICAM1 protein expression was analyzed by western blot analysis. ( g , h ) The relative levels of VCAM1 ( g ) and ICAM1 ( h ) mRNA were determined by qRT-PCR. All experiments were repeated three times ( n = 3), and data are presented as the mean ± S.E. *** p < 0.001 and ** p < 0.01 vs. untreated scrambled siRNA-transfected control cells. ### p < 0.001 and # p < 0.05 vs. TNF-α-treated scrambled siRNA-transfected cells.

Article Snippet: 3XFlag pCMV5-TOPO TAZ WT (Addgene plasmid # 24809) and 3XFlag pCMV5-TOPO TAZ (S89A) were a gift from Jeff Wrana (Addgene plasmid # 24815) (Varelas, Sakuma et al., 2008).

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR