kif2a  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: KIF2A dysfunction elicits genomic stability in BRCA1-deficient tumor cells. ( A ) Comparisons of the effect of each gene on the HRD effect between BRCA1-deficient ( n = 174) and BRCA1-sufficient ( n = 697) breast cancer samples. BRCA1 deficiency was defined as a BRCA1 mRNA expression level in the lowest 20% of BRCA1 mRNA levels. The y -axis and x -axis values of each point represent the HRD effect (the difference between the median HRD score of the high-expression samples and that of the low-expression samples) of each gene ( n = 8668) in BRCA1-deficient and BRCA1-sufficient breast cancers. We excluded genes that significantly affected the HRD score in the same way in both groups of samples stratified by the BRCA1 background. The circle size indicates the P -value of the HRD effect in BRCA1-deficient breast cancer samples. “BRCA1 synthetic rescue targets” (y > 0 and P -value <.05) are enclosed in the red box. The dashed line indicates the threshold (HRD effect in BRCA1-deficient BC > 18) for selecting top-ranking BRCA1 synthetic rescue targets. BC, breast cancer. ( B ) Analysis of the LOH, LST, TAI, and HRD scores in breast cancer samples from TCGA. Sample size ( n ): BRCA1_high&KIF2A_high n = 391; BRCA1_high&KIF2A_low n = 305; BRCA1_low&KIF2A_high n = 44; BRCA1_low&KIF2A_low n = 131. The center line indicates the median. The lower and upper hinges correspond to the first and third quartiles (25th and 75th percentiles), respectively. The upper and lower whiskers extend from the hinge to the largest and smallest values, respectively, no further than 1.5 × interquartile range from the hinge. Data beyond the end of the whiskers, considered “outlier” values, are plotted individually. ( C, D ) Metaphase spread of HeLa cells transfected with the indicated siRNAs for 48 h and treated with dimethyl sulfoxide (DMSO) or Olaparib for 16 h. Representative images are shown in panel (C). The statistical analysis results of the number of structural chromosomal abnormalities per metaphase are shown in panel (D). Metaphase number (n): siNC_DMSO n = 32; siBRCA1_DMSO n = 54; siBRCA1 + siKIF2A_DMSO n = 49; siNC_ Olaparib n = 49; siBRCA1_ Olaparib n = 44; siBRCA1 + siKIF2A_ Olaparib n = 46. ( E, F ) Viability of HeLa ( E ) and HCC1937 ( F ) cells transfected with the indicated siRNAs and treated with the indicated concentration of Olaparib, as determined by colony formation assays [mean ± standard deviation (SD), n = 3]. ( G ) Correlation of the KIF2A or KIF24 expression level and Olaparib sensitivity in breast (left) and ovarian (right) cancer cell lines in the CellMiner database. BR, breast; OV, ovarian. P -values were calculated with the Wilcoxon rank-sum test [panels (A), (B), and (D)], two-way analysis of variance (ANOVA) [panels (E) and (F)] and Pearson correlation analysis ( G ). ** P <.01; **** P <.0001.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Expressing, Transfection, Concentration Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: KIF2A is required for 53BP1 and RIF1 recruitment at DSB sites and for efficient NHEJ. ( A ) HR efficiency was analysed upon transfection of DR-GFP stable U2OS cells with the indicated siRNAs [mean ± standard error of the mean (SEM), n = 3]. ( B ) (Left) Representative images of RAD51 foci in HeLa cells transfected with the indicated siRNAs for 48 h and released 3 h after 10 Gy IR treatment. Scale bars, 10 µm. (Right) Statistical analysis of RAD51 foci per S/G2-phase cell. S/G2 nuclei number ( n ): siNC n = 108; siBRCA1 n = 163; siBRCA1 + siKIF2A n = 171. ( C ) pRPA2 (S4/S8) in HeLa cells transfected with the indicated siRNAs for 48 h and released 3 h after 10 Gy IR treatment. (Left) Representative images of pRPA2 (S4/S8) in pre-extracted cells. Scale bars, 10 µm. (Right) Statistical analysis of pRPA2 (S4/S8) intensity per nucleus. Nuclei number (n): siNC n = 60; siBRCA1 + siNC n = 69; siKIF2A n = 100; siBRCA1 + siKIF2A n = 100. ( D ) (Left) Representative images of 53BP1 foci in HeLa cells transfected with the indicated siRNAs for 48 h and released 1 h after 10 Gy IR treatment. Scale bars, 10 µm. (Right) Statistical analysis of the percentage of cells with ≥ 20 53BP1 foci (mean ± SEM, n = 3). ( E ) (Left) Representative images of RIF1 foci in HeLa cells transfected with the indicated siRNAs for 48 h and released indicated times after 3 Gy IR treatment. Scale bars, 10 µm. (Right) Statistical analysis of the number of RIF1 foci per nucleus. Nuclei number ( n ): siNC 1 h n = 157, 5 h n = 173, 9 h n = 193; siKIF2A 1 h n = 157, 5 h n = 172, 9 h n = 225. ( F–H ) The viability of HeLa cells transfected with the indicated siRNAs and treated with the indicated dose of IR ( F ), bleomycin ( G ), or Zeocin ( H ) was evaluated by a colony formation assay (mean ± SD, n = 3). ( I ) Statistical analysis of γH2AX intensity per nucleus in HeLa cells transfected with the indicated siRNAs for 48 h and released 1, 8, or 18 h after 3 Gy IR treatment. Nuclei number ( n ): siNC 1 h n = 381, 8 h n = 259, 18 h n = 274; siKIF2A 1 h n = 362, 8 h n = 266, 18 h n = 314; siBRCA1 + siNC 1 h n = 341, 8 h n = 342, 18 h n = 314; siBRCA1 + siKIF2A 1 h n = 380, 8 h n = 360, 18 h n = 319. ( J–L ) Representative images of metaphase spread of HeLa cells transfected with the indicated siRNAs for 72 h and treated with 1 Gy IR 5 h before collection ( J ). Statistical analysis of the number of chromosomal breaks per metaphase. Metaphase number ( n ): siNC n = 70; siKIF2A #1 n = 72; siKIF2A #2 n = 74; siKIF2A #3 n = 71 ( K ). Statistical analysis of the number of structural chromosomal abnormalities per 100 metaphases (mean ± SEM, n = 3) ( L ). ( M ) NHEJ efficiency in EJ5-GFP U2OS cells transfected with the indicated siRNAs (mean ± SEM, n = 3). ( N ) NHEJ efficiency in EJ5-GFP U2OS cells transfected with Tet-inducible pCW57-KIF2A plasmid and the indicated siRNAs, with or without treatment of 1.5 µg/ml DOX to induce KIF2A expression (mean ± SEM, n = 4). P -values were calculated with the paired two-tailed Student’s t -test [panels (A), (M), and (N)], the Wilcoxon rank-sum test [panels (B), (C), (E), (I), and K)], unpaired two-tailed Student’s t -test [panels (D) and (L)] or two-way ANOVA [panels (F)–(H)]. * P <.05; ** P <.01; *** P <.001; **** P <.0001; ns, not significant.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Transfection, Colony Assay, Plasmid Preparation, Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: NE remodelling mediated by microtubules in response to DNA damage depends on KIF2A. ( A ) Representative images of of HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Scale bars, 3 µm. ( B ) Representative orthogonal view images of NE with or without 10 Gy IR treatment and released at 6 h. Scale bars, 10 µm. Blue arrows: shallower wrinkle-like invaginations, yellow arrows: deeper tubule-like invaginations. ( C ) Percentage of HeLa cells with a wrinkled NE at 0, 1, or 6 h after 10 Gy IR treatment (mean ± SEM, n = 3). ( D ) Representative orthogonal view images of NE in HeLa cells transfected with siNC or siKIF2A and released 6 h after 10 Gy IR treatment. Scale bars, 10 µm. Blue arrows: shallower wrinkle-like invaginations, yellow arrows: deeper tubule-like invaginations. ( E ) Nuclear invagination index (2D) of HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): siNC n = 149; siKIF2A n = 150; siKIF2C n = 96. ( F ) Nuclear invagination index (3D) of HeLa cells transfected with siNC or siKIF2A at 0, 1, or 6 h after treatment with 10 Gy IR. Nuclei number ( n ): siNC 0 h n = 115, 1 h n = 110, 6 h n = 104; siKIF2A 0 h n = 125, 1 h n = 113, 6 h n = 91. ( G ) Nuclear invagination index (2D) of HCC1937 cells (left) transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): siNC n = 126; siKIF2A n = 130. Distribution of number of tubules per nucleus in MDA-MB-231 and U2OS cells (middle and right). Cells were transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment (mean ± SD, n = 7, 8). ( H ) Nuclear invagination index (2D) of G1- and S/G2-phase (classified based on the intensity of the Cyclin A2 signal) HeLa cells transfected with siNC or siKIF2A and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): G1_siNC n = 86; G1_siKIF2A n = 52; S/G2_siNC n = 83; S/G2_siKIF2A n = 99. ( I ) Immunofluorescence staining of HCC1937 cells treated with 5 Gy IR and released at 6 h. Scale bars, 10 µm. ( J ) The fluorescence intensities of the DAPI, KIF2A, or tyr-α-tubulin signals plotted against their distances from the blue dot lines in panel (I). ( K ) Super-resolution images of HeLa cells transfected with siNC or siKIF2A and treated with or without 5 µg/ml bleomycin for 6 h. Scale bars, 5 µm. ( L ) Nuclear invagination index (2D) of HeLa cells transfected with siNC or siKIF2A, pre-treated with 200 ng/ml nocodazole or 1 µM paclitaxel for 4 h and further treated with 10 Gy IR and released at 1 h. Nuclei number ( n ): DMSO_siNC n = 95; DMSO _siKIF2A n = 79; Nocodazole_siNC n = 98; Nocodazole_siKIF2A n = 82; Paclitaxel_siNC n = 91; Paclitaxel _siKIF2A n = 84. ( M ) Nuclear invagination index (3D) of HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): siNC + siNC n = 126; siNC + siKIF2A n = 100; siSUN1 + siNC n = 130; siSUN1 + siKIF2A n = 153; siSUN2 + siNC n = 121; siSUN2 + siKIF2A n = 123; siSYNE2 + siNC n = 116; siSYNE2 + siKIF2A n = 127. ( N ) Nuclear invagination index (2D) of HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): siNC + siNC n = 342; siNC + siKIF2A n = 206; siLMNA + siNC n = 156; siLMNA + siKIF2A n = 159; siLMNB1 + siNC n = 197; siLMNB1 + siKIF2A n = 206. ( O ) Percentage of HeLa cells with wrinkled NE under various DDR inhibitions. Cells were pre-treated with the indicated inhibitors (ATR: VE-821, 5 µM; ATM: KU55933, 10 µM; DNA-PK: NU-7441, 2 µM) for 2 h and then exposed to Zeocin (200 µg/ml) for 6 h where indicated (mean ± SEM, n = 3). P -values were calculated with unpaired two-tailed Student’s t test [panels (C), (G), and (O)] or Wilcoxon rank-sum test [panels (E)–(H) and (L)–(N)]. * P <.05; ** P <.01; *** P <.001; **** P <.0001; ns, not significant.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Transfection, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: KIF2A-promoted 53BP1 recruitment at DSB sites depends on the regulation of microtubule depolymerization, the LINC complex and lamin B1. ( A ) Mapping of the KIF2A domain important for 53BP1 recruitment. ( B ) Western blot analysis of pRPA2 (S4/S8) levels in inducible KIF2A-mutant HeLa cells. Cells were transfected with siNC or siKIF2A #3, treated with or without 1 µg/ml DOX to induce the indicated KIF2A mutants, and harvested 4 h after 10 Gy IR treatment. Labels on the right indicate the positions corresponding to full-length KIF2A and ΔNECK-KIF2A proteins. ( C, D ) Representative images of 53BP1 foci in HeLa cells transfected with siNC or siKIF2A #3 and reintroduced the indicated KIF2A mutants with 1.5 µg/ml DOX and released 1 h after 10 Gy IR treatment. Scale bars, 10 µm. ( C ) Statistical analysis of 53BP1 foci per nucleus are shown in panel (D). Nuclei number ( n ): siNC n = 137; siKIF2A #3 + vector n = 133; siKIF2A #3 + WT n = 164; siKIF2A #3+∆Neck n = 105; siKIF2A #3+∆ATPase n = 122; siKIF2A #3+∆NLS n = 85. ( E, F ) Representative images of 53BP1 foci in HeLa cells transfected with siNC or siKIF2A, pre-treated with 200 ng/ml nocodazole or 1 µM paclitaxel for 4 h and released 1 h after 10 Gy IR treatment. Scale bars, 10 µm. Statistical analysis of 53BP1 foci per nucleus are shown in panel (F). Nuclei number ( n ): DMSO_siNC n = 153; DMSO _siKIF2A n = 182; Nocodazole_siNC n = 178; Nocodazole_siKIF2A n = 175; Paclitaxel_siNC n = 196; Paclitaxel _siKIF2A n = 153. ( G ) Statistical analysis of 53BP1 foci per nucleus in HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): siNC + siNC n = 186; siNC + siKIF2A #2 n = 184; siNC + siKIF2A #3 n = 177; siLMNA + siNC n = 176; siLMNA + siKIF2A #2 n = 170; siLMNA + siKIF2A #3 n = 161; siLMNB1 + siNC n = 190; siLMNB1 + siKIF2A #2 n = 190; siLMNB1 + siKIF2A #3 n = 150. Representative images are in . ( H ) Statistical analysis of 53BP1 foci per nucleus in HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Nuclei number ( n ): siNC + siNC n = 186; siNC + siKIF2A #2 n = 184; siSUN1 + siNC n = 166; siSUN1 + siKIF2A #2 n = 176. P -values were calculated with Wilcoxon rank-sum test. * P <.05; ** P <.01; *** P <.001; **** P <.0001; ns, not significant.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Western Blot, Mutagenesis, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: KIF2A-mediated NE invagination shortens the distance between DSBs and NE to enhance 53BP1 recruitment. ( A ) Representative orthogonal view images of 53BP1 foci (green) and NE (red) in MDA-MB-231 cells transfected with siNC or siKIF2A and released 1 h after 10 Gy IR treatment. Scale bars, 10 µm. ( B ) Distance from 53BP1 foci to NE in HeLa cells transfected with siNC or siKIF2A released 1 h post 10 Gy IR, measured from 3D reconstructions. Foci number ( n ): siNC n = 582, siKIF2A n = 826. ( C ) Distance from 53BP1 foci to NE in HeLa Tet-inducible KIF2A-WT cells, following transfection with indicated siRNAs and DOX treatment (1.5 µg/ml, 48 h), and released 1 h post 10 Gy IR. Distance were measured from 3D reconstructions. Foci number ( n ): siNC n = 396, siKIF2A#3 n = 761; siKIF2A#3 + DOX n = 907. ( D ) Correlation of 53BP1 foci volume with minimum distance to NE in 3D reconstructed HeLa cells 1 h after 10 Gy IR treatment. Each data point corresponds to a single focus, and colors represent three different cells. The global Pearson correlation coefficient ( R ) and P -value are shown. Correlations for each cell separately are provided in . ( E ) GFP-53BP1 (tudor) recruitment to laser micro-irradiation sites in Hela cells. (Top) Live-cell images at indicated time points, captured at the bottom and middle focal planes. Laser positions are marked by yellow arrowheads. Scale bars, 10 µm. (Bottom) Quantified relative intensity of GFP-53BP1 (mean ± SEM, n = 8). Schematic diagram illustrating the position of the focal plane. ( F ) GFP-53BP1 (tudor) recruitment to laser stripes at interior versus perinuclear sites. (Top) Live-cell images at middle planes over time. Yellow arrowheads indicate laser tracks. Scale bars, 10 µm. (Bottom) Quantified relative intensity of GFP-53BP1 (tudor) (mean ± SEM, n = 3). P -values were calculated with the Wilcoxon rank-sum test [panels (B) and (C)], Pearson correlation analysis [panel (D)], and two-way ANOVA [panels (E) and (F)]. ** P <.01; **** P <.0001; ns, not significant.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Transfection, Irradiation

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: KIF2A binds to microtubules in response to DNA damage. ( A ) Western blot results showing the KIF2A and α-tubulin levels in the polymerized microtubule fraction and free tubulin fraction in HeLa cells subjected to the indicated treatments and released at 5 h. Paclitaxel was added for 5 h as a positive control for the MT fractionation assay. ( B, C ) Representative reconstructed images of NE surface and PLA (KIF2A and α-tubulin) foci in HeLa cells treated with DMSO, Zeocin (100 µg/ml), or bleomycin (5 µg/ml) for 6 h. Scale bars, 3 µm. The statistical analysis of the number of PLA foci per cell is shown in panel (C) . Cell number ( n ): NT n = 49; Zeocin n = 58; Bleomycin n = 77. ( D, E ) KIF2A immunofluorescence intensity (permeabilization before fixation) in HeLa cells treated with DMSO, IR (5 Gy, 6 h) or bleomycin (5 µg/ml, 6 h). The statistical analysis of the KIF2A intensity per cell is shown in panel (E). Cell number ( n ): DMSO n = 61; IR n = 73; Bleomycin n = 84. ( F, G ) Sum of the z-stack of the KIF2A immunofluorescence intensity (permeabilization before fixation) in the bottom plane in HeLa cells that were synchronized to G1/S phase and then treated with or without 200 µg/ml Zeocin for 6 h. Statistical analysis of the KIF2A intensity per cell is shown in panel (G). Cell number ( n ): DMSO n = 66; Zeocin n = 65. ( H, I ) Sum of the z-stack of the KIF2A immunofluorescence intensity (permeabilization before fixation) in HeLa cells pre-treated with an ATR inhibitor (VE-821, 5 µM), an ATM inhibitor (KU55933, 10 µM) or a DNA-PK inhibitor (NU-7441, 2 µM) for 2 h and then treated with or without Zeocin (200 µg/ml) for 6 h. Cell number ( n ): DMSO_NT n = 47; DMSO_Zeocin n = 46; ATMi_NT n = 28; ATMi _Zeocin n = 39; ATRi _NT n = 36; DMSO_ ATRi n = 33; DNA-PKi_NT n = 38; DNA-PKi _Zeocin n = 40. P -values were calculated with the Wilcoxon rank-sum test. * P <.05; ** P <.01; ***, P <.001; **** P <.0001; ns, not significant.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Western Blot, Positive Control, Fractionation, Immunofluorescence

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: α-Tubulin tyrosination drives KIF2A-mediated NE invagination upon DNA damage. ( A, B ) Immunofluorescence staining of HeLa cells treated with DMSO, IR (5 Gy, 6 h) or bleomycin (5 µg/ml, 6 h). Scale bar, 10 µm. The statistical analysis of the ratio of the intensity of tyrosinated α-tubulin to that of total α-tubulin per cell is shown in panel (B). Cell number ( n ): DMSO n = 52; IR n = 47; Bleomycin n = 36. ( C ) Statistical analysis of the ratio of the intensity of tyrosinated α-tubulin to that of total α-tubulin according to Western blot analysis (mean ± SEM, n = 3). ( D ) Statistical analysis of the ratio of the intensity of tyrosinated α-tubulin to that of total α-tubulin per cell in HeLa pre - treated with an ATM inhibitor (KU55933, 10 µM) and then treated with or without Zeocin (200 µg/ml) for 6 h. Cell number ( n ): DMSO_NT n = 79; DMSO_Zeocin n = 59; ATMi_NT n = 72; ATMi_Zeocin n = 77. ( E ) Statistical analysis of the ratio of the intensity of tyrosinated α-tubulin to that of total α-tubulin per cell in HeLa transfected with the indicated siRNAs and treated with or without IR (5 Gy, 6 h). Cell number ( n ): siNC_NT n = 87; siNC_IR n = 100; siTTL_NT n = 49; siTTL_IR n = 71; siVASH1_NT n = 69; siVASH1_IR n = 74. ( F ) Statistical analysis of relative KIF2A intensity (the ratio of the intensity of KIF2A to that of total α-tubulin) per cell in HeLa transfected with the indicated siRNAs and treated with or without IR (5 Gy, 6 h). Cell number ( n ): siNC_NT n = 131; siNC_IR n = 116; siTTL_NT n = 107; siTTL_IR n = 97; siVASH1_NT n = 82; siVASH1_IR n = 105. ( G , H ) Nuclear sphericity in HeLa cells transfected with the indicated siRNAs and released 1 h after 10 Gy IR treatment. Scale bars, 10 µm. Statistical analysis of nuclear sphericity is shown in panel (H) Nuclei number ( n ): siNC + siNC n = 126; siNC + siKIF2A n = 100; siVASH1 + siNC n = 156; siVASH1 + siKIF2A n = 95. P -values were calculated with unpaired two-tailed Student’s t -test [panels (B)–(E)] or the Wilcoxon rank-sum test [panels (F) and (H)]. * P <.05; *** P <.001; **** P <.0001; ns, not significant.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Immunofluorescence, Staining, Western Blot, Transfection, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: KIF2A-mediated microtubule-dependent nuclear envelope invagination drives nonhomologous end joining

doi: 10.1093/nar/gkag004

Figure Lengend Snippet: Microtubules facilitate NHEJ repair through KIF2A-mediated NE invagination. Models depicting microtubule dynamics, NE remodeling and 53BP1 recruitment following DNA damage in both KIF2A-WT cells (top) and KIF2A-deficient cells (bottom). In KIF2A-WT cells, DNA damage triggers ATM activation, leading to increased α-tubulin tyrosination. This post-translational modification enhances KIF2A binding to microtubules, promoting microtubule turnover and subsequent NE invagination through mechanisms dependent on the LINC complex and lamin B1. The resulting structural changes bring DSB sites closer to NE, facilitating efficient 53BP1 recruitment and promoting accurate NHEJ repair. In contrast, KIF2A-deficient cells exhibit defective microtubule dynamics despite normal ATM activation and α-tubulin tyrosination. The absence of functional KIF2A prevents NE invagination formation, maintaining an extended distance between 53BP1 foci and NE. This spatial disruption impairs 53BP1 accumulation at DSB sites and compromises NHEJ repair fidelity, resulting in increased repair errors.

Article Snippet: After blocking with 5% milk, the membrane were incubated with the following antibodies at 4°C overnight: Flag (1:5000, F3165, Sigma–Aldrich), RPA32/RPA2 (phospho S4 + S8) (1:2000, ab87277, Abcam), KIF2A (1:2500, 13105-1-AP, Proteintech), GAPDH (1:10 000, KM9002, Sungene), BRCA1 (1:1000, OP92, Millipore), 53BP1 (1:1000, 4937, Cell Signaling Technology), RIF1 (1:3000, A300-569A, Bethyl), KIF2C (1:100, sc-81305, Santa Cruz Biotechnology), α-tubulin (1:5000, T6199, Sigma–Aldrich), Lamin A/C (1:2500, 10298-1-AP, Proteintech), Lamin B1 (1:10 000, HRP-66095, Proteintech), SUN1 (1:5000, ab124770), SUN2 (1:5000, ab124916), SYNE2 (1:1000, ab314872), TTL (1:2000, 66076-1-Ig), VASH1 (1:500, 12730-1-AP), Pan-Ser/Thr (1:1000, AP1067, ABclonal), Tubulin antibody [YL1/2] (1:5000, ab6160, Abcam), or H3 (1:5000, B1055, Biodragon).

Techniques: Activation Assay, Modification, Binding Assay, Functional Assay, Disruption