Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: Monomeric MAD2L2 can bind to CDH1: ( A ) Schematic representation of MAD2L2 residues mutated to disrupt homodimerization. ( B ) Myc-2DD and myc-3DD mutants displayed reduced binding to YFP-MAD2L2. HEK293 cells were co-transfected with the indicated plasmids, and α-myc IP was performed to assess MAD2L2 homodimer formation. The binding of myc-MAD2L2 to YFP-MAD2L2 was reduced in all myc-MAD2L2 mutants when compared to WT. **IgL–IgG Light chain. ( C ) MAD2L2-CDH1 binding was reduced in the HA-3DD mutant. HEK293 cells were co-transfected with the indicated plasmids, and α-myc IP was performed to assess MAD2L2-CDH1 binding. The binding of myc-CDH1 to HA-MAD2L2 was reduced in HA-MAD2L2 3DD and HA-R124A mutants when compared to WT and HA-2DD. ( D ) The quantification of the relative amount of myc CDH1 bound to each HA-MAD2L2 mutant. Error bar = 1 SD. The additional blots contributing to this analysis are shown in . ( E ) HA-MAD2L2 A135D mutant displayed reduced binding to mRFP-CDH1. HEK293 cells were co-transfected with the indicated plasmids, and α-HA IP was performed to assess MAD2L2-CDH1 binding. The binding of HA-MAD2L2 A135D mutant to mRFP-CDH1 was reduced compared to WT. ( F ) The alignment of the SHLD3 RFIP motif to the proposed CDH1 RFIP motif, revealing conserved residues shared in both proteins. ( G ) HA-CDH1 F48A exhibited binding to MAD2L2. HEK293 cells were co-transfected with the indicated plasmids, and α-GFP IP was performed to assess MAD2L2-CDH1 binding. The binding of both HA-CDH1 WT and F48A was detected. ( H ) HA-CDH1 F48A binding to endogenous CDC27 (APC3) was reduced. HEK293 cells were co-transfected with HA CDH1 WT or F48A mutant, and α-HA IP was performed to assess CDC27 CDH1 binding.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Binding Assay, Transfection, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: Monomeric MAD2L2 can regulate mitotic entry: ( A ) Clones of U2OS CRISPR/Cas9 knockout mad2l2 cell line and the stably complemented mad2l2 #1 cell line with different HA-tagged MAD2L2 mutants, as indicated. ( B ) The quantification of the time taken for knockout U2OS mad2l2 and the complemented HA- mad2l2 cells to reach the metaphase–anaphase transition from nuclear envelope breakdown (NEBD), assessed by time-lapse video microscopy (1 frame every 5 min). The plot shows the cumulative percentage of cells that progressed to anaphase. “n” represents the number of cells examined, collected from at least three independent experiments. The p -value between U2OS and mad2l2 cell lines was calculated using a one-tailed t -test. ( C ) Percentage of anaphase bridges (upper panel) and lagging chromosomes (lower panel) in mad2l2 and the complemented HA- mad2l2 U2OS cell lines. “n” represents the number of anaphases examined; error bar = 1 SD; n.s-not significant; p -value between the indicated cell lines was calculated using a one-tailed t -test.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Clone Assay, CRISPR, Knock-Out, Stable Transfection, Microscopy, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: Monomeric MAD2L2 inhibits APC/C activation: ( A ) mad2l2 clones present premature binding of CDH1 to the APC/C. In order to assess the binding of CDH1 to the APC/C in each cell line, an α-CDC27 IP was performed on U2OS and mad2l2 clones that were arrested in nocodazole for 16h. In both mad2l2 clones, but not in WT cells, CDH1 prematurely binds to the CDC27 subunit of the APC/C, during nocodazol arrest. ( B ) mad2l2 :HA-MAD2L2 restores the canonical binding pattern of CDH1 to the APC/C and prevents premature binding of CDH1 to the APC/C. In order to assess the binding of CDH1 to the APC/C in each cell line, an α-CDC27 IP was performed on cells arrested in nocodazole for 16h. WT and mad2l2 :HA-MAD2L2 cells present lower binding to the CDC27 subunit, compared to mad2l2 cells. ( C ) mad2l2 :HA-MAD2L2 2DD is able to prevent the premature binding of CDH1 to the APC/C. In order to assess the binding of CDH1 to the APC/C in each cell line, an α-CDC27 IP was performed on cells arrested in nocodazole for 16 h. WT and mad2l2 :HA-MAD2L2 2DD present lower binding present lower binding to the CDC27 subunit, compared to mad2l2 cells. ( D ) Both mad2l2 :HA-MAD2L2 3DD and mad2l2 :HA-MAD2L2 A135D present premature binding of CDH1 to the APC/C and fail to restore the normal binding pattern, as indicated by the relatively high level of CDH1 bound to CDC27 during nocodazole treatment. **IgL–IgG Light chain. ( E ) The quantification of the relative amount of CDH1 bound to CDC27 in all presented cell lines. Each dot represents an independent repeat of the IP. The blots contributing to this analysis are shown in –E. ( F ) Premature binding of CDH1 to the APC/C, during nocodazole arrest, leads to a reduction in CDH1 levels. Endogenous levels of CDH1 were monitored in the indicated cell lines after 16 h of nocodazole treatment. Lack of MAD2L2, or the presence of MAD2L2 without the ability to bind CDH1, leads to premature CDH1 degradation. ( G ) The quantification of the relative amount of endogenous CDH1 in nocodazole-arrested cells. Each dot represents an independent repeat. Blots contributing to this analysis are shown in . ( H ) Premature binding of CDH1 to the APC/C, during nocodazole arrest, leads to a reduction in Aurora A levels. Endogenous levels of Aurora A were monitored in the indicated cell lines after 16h of nocodazole treatment. Lack of MAD2L2, or the presence of MAD2L2 without the ability to bind CDH1, leads to premature Aurora A degradation. ( I ) The quantification of the relative amount of endogenous Aurora A in nocodazole-arrested cells. Each dot represents an independent repeat. The additional blots contributing to this analysis are shown in . For all: Error bar = 1 SD; p -value was calculated using a one-tailed t -test.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Activation Assay, Clone Assay, Binding Assay, One-tailed Test

Journal: International Journal of Molecular Sciences

Article Title: MAD2L2 Dimerization Is Not Essential for Mitotic Regulation

doi: 10.3390/ijms252111485

Figure Lengend Snippet: CDH1 overexpression reduces MAD2L2 homodimerization: ( A ) A graphic model presenting two options for MAD2L2-CDH1 interaction. Option 1: CDH1 can bind both monomeric and dimeric forms of MAD2L2 via the L186 residue. Option 2: CDH1 binds to the monomeric form of MAD2L2 using both L186 and A135 residues, blocking MAD2L2 homodimerization. ( B ) In situ MAD2L2 homodimer formation is reduced upon CDH1-GFP overexpression, as indicated by a reduced number of foci/cell. Representative images of the PLA foci. ( C ) The quantification of MAD2L2 homodimer from the PLA assay; error bar = 1 SD; each dot represents a cell. ( D ) The number of foci/cell for each cell line in a cumulative percentage plot to observe the difference between cell populations upon treatments; p -value was calculated using a one-tailed t -test.

Article Snippet: Guide RNA sequences targeting MAD2L2 were phosphorylated, annealed, and ligated into a pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (#42230, Addgene, a gift from Dr. Gabi Gerlitz Ariel University).

Techniques: Over Expression, Residue, Blocking Assay, In Situ, One-tailed Test

Journal: The EMBO Journal

Article Title: Synergistic and antagonistic activities of IRF8 and FOS enhancer pairs during an immune-cell fate switch

doi: 10.1038/s44318-025-00380-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The base px330 plasmid was a gift from Jinsong Li (Addgene plasmid # 98750; http://n2t.net/addgene:98750 ; RRID: Addgene_98750 ).

Techniques: Recombinant, Clone Assay, Binding Assay, Sequencing, CRISPR, Magnetic Beads, Software, Nick Translation, Electron Microscopy, Plasmid Preparation, DNA Extraction, Reverse Transcription, Purification