Journal: Nucleic Acids Research

Article Title: REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ

doi: 10.1093/nar/gku1385

Figure Lengend Snippet: REV7 interacts with full-length REV3L in vivo. ( A ) Schematic of REV3L, showing the locations of the evolutionarily conserved N-terminal domain, the B-family DNA polymerase domain and the previously proposed REV7 binding site. An iron-sulfur cluster in the Cys-rich domain near the C-terminus provides a binding site for the POLD2 subunit. The position of FLAG and HA tags is shown. ( B ) Human 293T cells were transfected with either epitope-tagged REV3L (FH-REV3L) or Flag-HA empty vector (Control). Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with FLAG antibody beads. After electrophoretic transfer of proteins, the membrane was cut into three sections to separate proteins >250 kDa (FLAG), 37 to 250 kDa (HA and α-tubulin) and <37 kDa (REV7) and immunoblotted with the indicated antibodies. Results for the input and IP product after gel electrophoresis are shown. ( C ) FLAG-IP experiment similar to part A, using FH-REV3L, FH-REV3L mutant construct (P1880A, P1885A) and control empty vector.

Article Snippet: Assay ID was Hs01057448_m1.

Techniques: In Vivo, Binding Assay, Transfection, Plasmid Preparation, Control, Immunoprecipitation, Membrane, Nucleic Acid Electrophoresis, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ

doi: 10.1093/nar/gku1385

Figure Lengend Snippet: Identification of a novel in vivo REV7 binding region in REV3L. ( A ) Schematic showing vectors expressing FH-REV3L amino acid residues 1974–3130 and 2000–3130. ( B ) FLAG-IP of 293T cells transfected with FH-REV3L 1974–3130, FH-REV3L 2000–3130. The bottom panels show low intensity and high intensity exposures. IgG light chain migrating slightly above REV7 is indicated by the asterisk (*). ( C ) Sequence alignments of a region of REV3L, showing the original REV7 and newly identified REV7 binding sites, with the consensus sequence ϕϕxPxxxpPSR at the bottom. Numbers refer to human REV3L residues. ( D ) Schematic drawing showing locations of the REV3L fragments. The P1996/P2001 position is highlighted with a vertical bar (green in the online version). ( E ) Immunopurification of GFP fusion fragments of REV3L with anti-GFP agarose. Immunoblotting used anti-FLAG, anti-GFP, anti-REV7 and anti-αTubulin.

Article Snippet: Assay ID was Hs01057448_m1.

Techniques: In Vivo, Binding Assay, Expressing, Transfection, Sequencing, Immu-Puri, Western Blot

Journal: Nucleic Acids Research

Article Title: REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ

doi: 10.1093/nar/gku1385

Figure Lengend Snippet: A novel REV7 binding site in REV3L. ( A ) In vitro association of REV7 with fragments of REV3L. GST fusion fragments of REV3L and His-REV7 fusion protein were purified from Escherichia coli (see Supplementary Figure S1B). These were used with glutathione beads for GST pulldown experiments. After electrophoresis, samples were immunoblotted with anti-His or anti-GST as indicated. The positive control REV3L 1847–1898 fragment included the original REV7 binding site (P1880, P1885); the negative control REV3L 1974–1999 fragment is unable to bind REV7 (Figure ). ( B ) In vitro GST pulldown of purified REV3L fragments (see Supplementary Figure S1C) containing the indicated amino acid changes. ( C ) GFP-REV3L fusion fragments (and mutant versions) were expressed in human 293T cells (Input) and then immunoprecipitated with anti-GFP (IP). Following gel electrophoresis, immunoblotting was performed with the indicated antibodies. ( D ) Schematic of REV7 binding sites in REV3L. ( E ) FLAG-IP assay of 293T cells transfected with FH-REV3L, FH-REV3L (P1880A, P1885A), FH-REV3L (P1880A, P1885A, P1996A, P2001A), FH-REV3L (P1996A, P2001A) or control empty vector. For immunoblotting, we used anti-HA, anti-REV7 and anti-αTubulin.

Article Snippet: Assay ID was Hs01057448_m1.

Techniques: Binding Assay, In Vitro, Purification, Electrophoresis, Positive Control, Negative Control, Mutagenesis, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot, Transfection, Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ

doi: 10.1093/nar/gku1385

Figure Lengend Snippet: Both REV7 binding sites in REV3L are important for cellular resistance to DNA damage. ( A and B ) Expression of human REV3L rescues cisplatin and UVC sensitivity of Rev3L -null MEFs. The survival of three clones was monitored following cisplatin and UVC treatment. +/− vector (cl 1): a control Rev3L +/− subclone containing pOZN-empty vector (square symbols, blue line). −/− vector (cl 22): a Rev3L −/− knockout subclone containing pOZN-empty vector (triangle symbols, red line). −/− WT (cl H5) : a Rev3L −/− subclone containing pOZN- WT REV3L expression vector (circle symbols, green line). Quantitative RT-PCR with primers specific to human REV3L verified that human REV3L was expressed in the MEFs. MEF cell line clones stably expressing the indicated REV3L constructs were plated and exposed to the indicated doses of cisplatin ( C ) or UVC ( D ). Cellular viability was measured 48 h later. +/− vector (cl 1): circle symbols, blue line. −/− vector (cl 22): square symbols, red line. −/− 4A (cl 20) and cl 35 : a Rev3L −/− subclone containing pOZN-REV3L P1880A, P1885A, P1996A, P2001A expression vector (triangle symbols, purple line). ( E ) Rev3L -null MEFs stably expressed 4A-mutant REV3L at a ∼3-fold higher levels than Rev3L -null MEFs complemented with WT REV3L. Gapdh was used as an internal control. ( F ) REV3L mutants that impair REV7 binding are unable to rescue chromosomal instability. +/− vector (cl 1, 2, 14, 26): Rev3L +/− subclones containing pOZN-empty vector; −/− vector (cl 2, 12, 20): Rev3L −/− subclones containing pOZN-empty vector; −/− 4A (cl 20, 35): Rev3L −/− subclones containing pOZN-4A binding mutant. Infections of clones were done independently and repeated on different days. Cells were fixed and stained with DAPI and micronuclei were enumerated for at least three independent experiments per clone. Data represent mean ± SEM. (*) P < 0.05 by unpaired t -test.

Article Snippet: Assay ID was Hs01057448_m1.

Techniques: Binding Assay, Expressing, Clone Assay, Plasmid Preparation, Control, Knock-Out, Quantitative RT-PCR, Stable Transfection, Construct, Mutagenesis, Staining

Journal: Nucleic Acids Research

Article Title: REV7 is essential for DNA damage tolerance via two REV3L binding sites in mammalian DNA polymerase ζ

doi: 10.1093/nar/gku1385

Figure Lengend Snippet: Model employing for promotion of functional interactions of REV7 by two binding sites in REV3L. ( A ) Two binding sites for REV7 on REV3L (vertical bars) would stabilize a homodimer of REV7. This would be functionally useful because REV7 has multiple binding partners (represented generically as ‘REV7 binding protein’ and REV7 interacts with two sites on REV1. In addition, some REV7 binding proteins may bind to the same region of REV7, so that two molecules of REV7 would be required to facilitate simultaneous interactions with multiple proteins. ( B and C ) Inactivation of one of the REV7 binding sites (black open bars) would still allow a monomer of REV7 to bind REV3L, but prevent the stable interaction with REV1 or other REV7 binding proteins. CTD, C-terminal domain. Black horizontal bar, Dimerization.

Article Snippet: Assay ID was Hs01057448_m1.

Techniques: Functional Assay, Binding Assay

Journal: Cell reports

Article Title: KIF18A promotes chromosome congression in cooperation with CENP-E downstream of CENP-C

doi: 10.1016/j.celrep.2025.116515

Figure Lengend Snippet: (A) Cell viability of K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days after doxycycline (Dox) addition (mean and SD, two-tailed Student’s t test, CENP-C WT cells: n = 6; CENP-C ΔM12BD cells: n = 6; ** p < 0.01). (B) Representative images of DAPI-stained K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Scale bar, 50 μm. (C) Population of normal interphase cells, mitotic cells, and cells with micronuclei in K562 WT or CENP-C ΔM12BD cells after knockout of indicated genes at 4 days. Error bars indicate SEM. n = 3 independent experiments; 200 cells from each cell line were quantified in each experiment. (D) The growth curve of K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. K562 WT or CENP-C ΔM12BD cells were treated with or without Dox ( KIF18A OFF or ON). The cell numbers were normalized to those at time 0 of each line. (E) Cell-cycle distribution of conditional knockout of KIF18A in K562 WT cells at each day after Dox addition, based on FACS analysis. (F) Cell-cycle distribution of conditional knockout of KIF18A in K562 CENP-C ΔM12BD cells at each day after Dox addition, based on FACS analysis. (G and H) Quantification of cells with misaligned chromosomes in K562 WT or CENP-C ΔM12BD cells with or without KIF18A knockout. The experimental scheme is shown. Cells were stained with antibodies against MAD2 (red) to detect misaligned chromosomes and CENP-T (green) as a kinetochore marker. DNA was stained with DAPI (blue). Arrowheads show typical MAD2-positive unaligned chromosomes. Scale bar, 10 μm. The cells with MAD2-positive chromosomes were quantified (H) (mean and SEM, two-tailed Student’s t test; n = 5 independent experiments; n.s., non-significant; ** p < 0.01). (I) Numbers of MAD2 positive kinetochores in each cell in each condition (WT KIF18A ON; WT KIF18A OFF; CENP-C ΔM12BD KIF18A ON; CENP-C ΔM12BD KIF18A OFF) (Mean and SEM, n = 5 independent experiments).

Article Snippet: Mouse anti-human MAD2 , Santa Cruz Biotechnology , Cat#sc-65492; RRID: AB_831526.

Techniques: Knock-Out, Two Tailed Test, Staining, Marker

Journal: Cancer Science

Article Title: Nucleoporin Nup188 is required for chromosome alignment in mitosis

doi: 10.1111/cas.12159

Figure Lengend Snippet: Nup188 depletion causes chromosome misalignment. (a) Depletion of Nup188 with a siRNA. Total cell lysates prepared from HeLa cells treated with mock or Nup188 siRNA for the indicated periods were separated by SDS‐PAGE and probed using western blotting with anti‐Nup188 or anti‐actin antibody. (b) Increased mitotic index in Nup188‐depleted cells. The mitotic index for mock‐, Nup188 siRNA‐ or Nup188/Mad2 siRNA‐treated HeLa cells was measured 48 h after transfection. Error bars represent the SD. (c) Nup188‐depleted cells showed chromosome misalignment. HeLa cells treated with mock or Nup188 siRNA for 48 h were stained with an anti‐tubulin antibody (green). DNA was stained with DAPI (blue). Bar, 10 μm. (d) Quantitative analysis of chromosome misalignment in Nup188‐depleted cells. HeLa cells were treated with mock or Nup188 siRNA for 48 h and with MG132 (10 μM) for the final 2 h. The number of misaligned chromosomes per cell was scored. Error bars represent the SD. (e) Mad2 localizes to kinetochores in Nup188‐depleted cells. HeLa cells treated with mock or Nup188 siRNA for 48 h were stained with an anti‐Mad2 antibody (green) and a CREST serum (red). Bar, 10 μm.

Article Snippet: The following polyclonal rabbit antibodies were used: NUP188 (A302‐322A; Bethyl Laboratories, Montgomery, TX, USA); Mad2 (Novus Biologicals, Littleton, CO, USA); Aurora A (ab12875; abcam, Cambridge, UK); and NuMA (A301‐509A; Bethyl Laboratories).

Techniques: SDS Page, Western Blot, Transfection, Staining

Journal: Molecular medicine reports

Article Title: Deficiency of PTEN leads to aberrant chromosome segregation through downregulation of MAD2.

doi: 10.3892/mmr.2019.10668

Figure Lengend Snippet: Figure 3. MAD2 is downregulated in PTEN‑deficient cells. (A) MAD2 expression was evaluated using cell lysates from control and PTEN‑knockdown cells by western blot analysis. (B) The protein expression levels of MAD2 and cyclin B1 in control and PTEN‑knockdown HeLa cells cultured with or without nocodazole were detected by western analysis. (C) Control and PTEN‑knockdown HeLa cells cultured with or without CHX were harvested at 0, 3 and 6 h after treatment. Then, the protein expression levels of MAD2 were evaluated using western blot analysis. (D) Control and PTEN‑knockdown HeLa cells cultured with or without MG132 were analyzed for MAD2 expression by western blot analysis. (E) Ubiquitin was overexpressed in control and PTEN‑knockdown HeLa cells. Following MG132 treatment, the cells were harvested for analysis via a ubiquitination assay. All data were obtained from 3 independent experi- ments. *P<0.05, **P<0.01 and ***P<0.001. MAD2, mitotic arrest deficient 2; PTEN, phosphatase and tensin homolog; sh, short hairpin RNA; Ub, ubiquitin; CHX, cycloheximide; IP, immunoprecipitates; IB, immunoblot.

Article Snippet: The following primary antibodies were used: rabbit anti-human PTen (1:2,000; cell Signaling Technology, Inc.; cat. no. 9188L), rabbit anti‐human MAD2 (1:1,000) and cyclin B1 (1:4,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc‐28261 and sc‐752, respectively), and mouse anti‐GAPDH (1:20,000; ProteinTech Group, Inc.; cat. no. 60004‐1‐Ig) were used.

Techniques: Expressing, Control, Western Blot, Cell Culture, Ubiquitin Proteomics, shRNA

Journal: Molecular medicine reports

Article Title: Deficiency of PTEN leads to aberrant chromosome segregation through downregulation of MAD2.

doi: 10.3892/mmr.2019.10668

Figure Lengend Snippet: Figure 4. Recovery of MAD2 expression partially ameliorates impaired mitosis. (A) PTEN‑knockdown HeLa cells were transfected with His‑tagged MAD2 expression plasmids prior to the analysis of MAD2 and cyclin B1 expression by western blot analysis. (B) The rate of cell survival for control, PTEN‑knockdown and His‑MAD2‑overexpression PTEN‑knockdown HeLa cells incubated with nocodazole for 36 h was calculated. (C) (Left panel) Representative images indicating the general morphology of mononucleated, binucleated and multinucleated cells at high magnification. (Middle panel) Representative images demonstrating cells of the shControl, shPTEN and shPTEN‑MAD2 groups stained by DAPI. Binucleated cells (arrowhead) and multinucleated cells (arrow) are marked in each image. (Right panel) Statistical analysis of the percentage of binucleated or multinucleated cells in the different groups. A total of 500 cells were counted. Among these cells, binucleated or multinucleated cells were counted. The percentage was calculated as binucleated cell number (or multinucle- ated cell number)/total cell number. All data were obtained from 3 independent experiments, *P<0.05, **P<0.01 and ***P<0.001. BF, bright field; MAD2, mitotic arrest deficient 2; PTEN, phosphatase and tensin homolog; sh, short hairpin RNA; his‑MAD2, His‑tagged MAD2 expression plasmid.

Article Snippet: The following primary antibodies were used: rabbit anti-human PTen (1:2,000; cell Signaling Technology, Inc.; cat. no. 9188L), rabbit anti‐human MAD2 (1:1,000) and cyclin B1 (1:4,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc‐28261 and sc‐752, respectively), and mouse anti‐GAPDH (1:20,000; ProteinTech Group, Inc.; cat. no. 60004‐1‐Ig) were used.

Techniques: Expressing, Transfection, Western Blot, Control, Incubation, Staining, shRNA, Plasmid Preparation