human kif2c  (Addgene inc)

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: TBX15/miR-152 pathway directly targets and inhibits KIF2C. A Western blotting analysis for protein expression of KIF2C in MCF7/ADR cells and T47D/ADR cells transfected with miR-152 mimics or miR-152 inhibitor, or TBX15-pCMV or siTBX15. All cell groups were treated with 1 µg/ml DOX. B Putative seed-matching sites or mutant sites (red) between miR-152 and the 3’-UTR of KIF2C were analyzed by TargetScan. The WT and mutant (mut) binding site sequences of miR-152 in the 3’ UTR of KIF2C were cloned into luciferase reporter vectors. HEK293T cells were co-transfected with the reporter vectors, renilla luciferase vector, and miR-152 mimics or miR-NC. After 24 h, the relative levels of luciferase activity were analyzed and normalized to values obtained from WT cells transfected with miR-NC. C Relative expression levels of KIF2C from normal tissue and breast tumors from human patients. KIF2C mRNA expression data were obtained from TCGA database containing 1114 breast tumors and 98 normal adjacent tissues, and were presented as the means ± SEM from three independent experiments. GSEA analysis to evaluate the correlation between expression levels of KIF2C and DOX resistance signatures in the GEO database. D MCF7/ADR cells were transfected with pCMV or KIF2C-pCMV plasmids (KIF2C), siNC or siKIF2C and were treated with DOX. After 48 h, drug sensitivity analysis was determined. E Flow cytometric analysis of MCF7/ADR cells which were transfected as in D and treated with 1 µg/ml DOX. F , G MCF7/ADR cells were transfected as above and were treated with 1 µg/ml DOX. After 48 h, the glucose consumption ( F , top), LA production ( F , bottom), and protein expression levels of KIF2C, p62, LC3, β-actin ( G ) were analyzed. All the data are presented as the means±SEM from three independent experiments and were normalized with corresponding control. *, ** P < 0.05 and P < 0.01, respectively

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Western Blot, Expressing, Transfection, Mutagenesis, Binding Assay, Clone Assay, Luciferase, Plasmid Preparation, Activity Assay, Control

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: KIF2C overexpression reverses miR-152-induced DOX sensitivity in MCF7/ADR cells. A Levels of KIF2C in MCF7/ADR cells which were co-transfected with miR-NC, miR-152 mimics, or KIF2C. The data are presented as the means±SEM from three independent experiments. * Compared to miR-NC group at P < 0.05, ## compared to miR-152 at P < 0.01. B IC50 values of MCF7/ADR cells that were transfected as in A and were treated with DOX and cultured for 48 h. C Flow cytometric analysis of MCF7/ADR cells that were transfected as in (A) and were treated with 1 µg/ml DOX. D , E The glucose consumption ( D , left), LA production ( D , right), ECAR) ( E , left) and OCR ( E , right) were analyzed in MCF7/ADR cells treated as in A . F Levels of KIF2C, p62 and LC3 in MCF7/ADR cells treated as in ( A ). Graph represents the relative density of KIF2C, p62, or LC3-II/LC3-I expression. The data are presented as the means ± SEM from three independent experiments. ** Compared to miR-NC group at P < 0.01, ## compared to miR-152 at P < 0.01. G The glucose consumption ( G , top) and LA production ( G , bottom) were analyzed in MCF7/ADR cells treated with TBX15 or KIF2C overexpression. H Flow cytometric analysis of MCF7/ADR cells that were transfected as in G and were treated with 1 µg/ml DOX. I Levels of p62 and LC3 in MCF7/ADR cells treated as in G . Graph represents the relative density of KIF2C, p62, or LC3-II/LC3-I expression. The data are presented as the means ± SEM from three independent experiments. *, ** Compared to control groups at P < 0.05 and P < 0.01, # compared to TBX15 at P < 0.05

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Over Expression, Transfection, Cell Culture, Expressing, Control

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: PKM2 is involved in DOX resistance in breast cancer, mediated by the miR-152/KIF2C signaling pathway. A Western blotting analysis of PKM2 levels in MCF7/ADR cells that were transfected with pCMV, TBX15, siNC or siTBX15, and were treated with 1 µg/ml DOX for 48 h. β-actin served as an internal control. B Western blotting analysis of PKM2 levels in MCF7/ADR cells transfected with KIF2C or siKIF2C, and were treated with 1 µg/ml DOX for 48 h. A , B Graph represents the relative density of PKM2 expression. β-actin was used as an internal control. The data are presented as the means ± SEM from three independent experiments.** Indicates significant difference compared to pCMV group at P < 0.01, # , ## compared to siNC at P < 0.05, P < 0.01, respectively. C Western blotting analysis of PKM2 levels in MCF7/ADR cells transfected with TBX15 and /or KIF2C, and were treated with 1 µg/ml DOX for 48 h. β-actin was used as an internal control. Graph represents the relative density of PKM2 expression. *,** Indicate significant difference compared to pCMV group at P < 0.05 and P < 0.01, respectively. # Compared to TBX15 at P < 0.05. D Western blotting analysis of PKM2 in MCF7/ADR cells that were co-transfected with miR-NC, miR-152 or KIF2C and were treated with 1 µg/ml DOX for 48 h. β-actin was used as an internal control. * Indicates significant difference compared to miR-NC group at P < 0.05, # compared to miR-152 at P < 0.05. E Western blotting analysis of KIF2C and PKM2 in MCF7/ADR cells that were co-transfected with siNC, siKIF2C or PKM2-pcDNA, and were treated with 1 µg/ml DOX for 48 h. Graph represents the relative density levels of PKM2, p62, or LC3-II/ I expression. β-actin was used as an internal control. ** Indicates significant difference compared to siNC group at P < 0.01, ## compared to siKIF2C at P < 0.01

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Western Blot, Transfection, Control, Expressing

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: KIF2C directly binds to PKM2 and promotes PKM2 stability in breast cancer cells. A Co-IP analysis of MCF7/ADR cells that were transfected with pCMV, TBX15, siNC or siTBX15 and were treated with 1 µg /ml DOX for 48 h. B Co-IP analysis of MCF7/ADR cells that were transfected with pCMV, KIF2C, siNC or siKIF2C. Cell lysates were co-immunoprecipitated using an anti-KIF2C or anti-PKM2 antibody. C Western blotting analysis of MCF7/ADR cells transfected with pCMV or KIF2C and treated with 20 µg /ml CHX for 0, 6, 12 and 18 h. D Western blotting analysis of MCF7/ADR cells transfected with siNC or siKIF2C and were treated with CHX. The expression levels of KIF2C and PKM2 were analyzed. E Western blotting analysis of MCF7/ADR cells transfected with a pCMV or KIF2C and treated with 1 µM MG132 for 0, 12 and 24 h. F Western blotting analysis of MCF7/ADR cells transfected with siNC or siKIF2C and treated with MG132. The expression of KIF2C and PKM2 were analyzed. G Western blotting analysis of MCF7/ADR cells transfected with pCMV, KIF2C, siNC or siKIF2C and Ubiquitin-HA plasmid. Cell lysates were co-immunoprecipitated using anti-HA antibody and the expression levels of Ub-PKM2 were analyzed. H Western blotting analysis of MCF7/ADR cells transfected with a pCMV, KIF2C, and KIF2C domains (D1, D2, D3) in pCMV-flag plasmids. Cells were treated with 1 µg /ml DOX for 48 h. Cell lysates were co-immunoprecipitated using anti-flag antibody and the expression levels of PKM2 were analyzed. I Western blotting analysis of MCF7/ADR cells that were transfected as ( H ), and Ubiquitin-HA plasmid. Cell lysates were co-immunoprecipitated using an anti-HA antibody and the expression levels of Ub-PKM2 were analyzed. All the data above were presented as the means ± SEM from three independent experiments and were normalized to the percentage of corresponding control groups. *, ** P < 0.05, P < 0.01, respectively

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Ubiquitin Proteomics, Plasmid Preparation, Control

Journal: Cancer Cell International

Article Title: TBX15/miR-152/KIF2C pathway regulates breast cancer doxorubicin resistance via promoting PKM2 ubiquitination

doi: 10.1186/s12935-021-02235-w

Figure Lengend Snippet: TBX15/miR-152 overexpression reverses DOX resistance in vivo by decreasing KIF2C and PKM2 expression. A–C The MCF7/ADR cells expressing miR-152 or a control (miR-NC) were established using a lentivirus system and dispersed in 100 µl of serum-free DMEM medium. Cells were subcutaneously injected into female nude mice (n = 8). After subcutaneous injection of cancer cells for 24 days, mice were treated with DOX or PBS. Representative pictures of tumors ( A ), tumor size ( B ), and weight ( C ) were obtained at the indicated time point or following 45 days after initial tumor cell injection. Tumors were measured every two days and the tumor volumes were calculated. The tumors were excised and weighed after 45 days, and the representative pictures of trimmed tumors are displayed (Bar= 10 mm). Data are presented as the means ± SEM from all tumor samples. D , E Immunohistochemistry staining of tumor tissues with antibodies of KIF2C and PKM2 (bar = 100 μm). * P < 0.05, ** P < 0.01. F , J The MCF7/ADR cells overexpressing TBX15 or a Negative control (NC) were established using lentivirus system. Cells were subcutaneously injected into female nude mice (n = 8). 10 days after cancer cells subcutaneous injection, tumor xenografts were found in ADR-NC group. However, only 4 tumor samples were found 31 days after injection in TBX15 overexpression group ( F ). Tumors growth was measured as above. The tumors were excised and weighed, and the representative pictures of trimmed tumors were displayed (Bar= 10mm). Data were presented as the means ± SEM from all tumor samples ( G , H ). I qRT-PCR analysis of miR-152 gene expression in ADR-NC and ADR-TBX15 tumor tissues. Data were normalized to the ratio of miR-152 in ADR-NC group. Data are presented as the means± SEM from all tumor samples. ** P < 0.01. J Immunohistochemistry staining of tumor tissues with antibodies of KIF2C and PKM2 (Bar= 100 μm in Low Power view and Bar= 50 μm in High Power view)

Article Snippet: The pCMV-based plasmid encoding human KIF2C or TBX15 and pcDNA3-based plasmid encoding human PKM2 were obtained from the nonprofit plasmid repository, Addgene (Cambridge, MA USA).

Techniques: Over Expression, In Vivo, Expressing, Control, Injection, Immunohistochemistry, Staining, Negative Control, Quantitative RT-PCR, Gene Expression

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) Beads conjugated with a biotin-double stranded DNA fragment (dsDNA, 500 bp, as described in Materials and methods—DNA binding assay) were incubated in Xenopus egg extracts for 30 min, re-isolated, and resolved by SDS-PAGE. The input, control pull-down (with blank beads), and biotin-dsDNA pull-down were analyzed by immunoblotting. ( B ) Beads conjugated with biotin-dsDNA (as in panel A) were incubated in HeLa cell lysates for 30 min, re-isolated, and resolved by SDS-PAGE. The input, control pull-down (with blank beads), and biotin-dsDNA pull-down were analyzed by immunoblotting. ( C ) Xenopus Kif2C was expressed with MBP-tag, and purified on amylose beads. As described in Materials and methods—pull-down assay, MBP-Kif2C or control (blank) beads were incubated in Xenopus egg extracts supplemented with cut or uncut plasmid, re-isolated, and analyzed by PCR and agarose gel electrophoresis/ethidium bromide staining. ( D ) As described in Materials and methods—pull-down assay, human Kif2C was expressed with MBP-tag and purified on amylose beads. MBP-Kif2C or control (blank) beads were incubated in the lysates of doxorubicin-treated HeLa cells. Pull-down samples were analyzed by mass spectrometry. The identified DNA repair proteins and numbers of peptides are shown. ( E ) GFP-Kif2C was expressed in HeLa cells with doxorubicin-treatment. Immunoprecipitation (IP) was performed using anti-GFP or control (blank) beads. 10% input, control and GFP IP samples were analyzed by immunoblotting.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: DNA Binding Assay, Incubation, Isolation, SDS Page, Control, Western Blot, Purification, Pull Down Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Mass Spectrometry, Immunoprecipitation

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) MBP-Kif2C pull down was performed in HeLa cells with or without doxorubicin (2 μg/mL) treatment. Input, control pulldown with blank beads, and MBP pulldown samples were analyzed by immunoblotting. ( B ) Doxorubicin treatment, as in panel A, activated DNA damage signaling, as indicated by Chk1 phosphorylation at Ser-317. ( C ) MBP-Kif2C pull down was performed in HeLa cells as in panel A. Cell lysates were incubated with DNase I (100 units/mL) as indicated. Input, control pulldown with blank beads, and MBP pulldown samples were analyzed by immunoblotting. ( D,E ) The N, M, C segments of Kif2C, as shown in panel D, were used for pull-down in the lysates of HeLa cells treated with doxorubicin. Control (ctr) pull-down was performed using blank beads. Immunoblots are shown in panel E.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Control, Western Blot, Phospho-proteomics, Incubation

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) HeLa cells expressing GFP-Kif2C were subjected to laser micro-irradiation as described in Materials and methods—immunofluorescence and imaging. The fluorescent signal of GFP is shown at the indicated time points. ( B ) The intensity of the GFP signal at laser-cut sites was normalized to that outside of the laser-cut sites for the relative enrichment of GFP-Kif2C. The mean values and standard deviations are shown (quantification shown in 5 cells, consistent pattern observed in >10 cells and >3 independent experiments). ( C ) HeLa cells expressing GFP-Kif2C were treated with 10 Gy IR, the fluorescent signal of GFP and immunofluorescent signal of γ-H2AX are shown. Pre-extraction was performed by placing the dish on ice for 5 min with 0.1% Triton X-100 in 10 mm HEPES (pH 7.4), 2 mm MgCl2, 100 mm KCl, and 1 mm EDTA. ( D ) GFP-Kif2C was expressed in HeLa cells. Prior to laser-micro-irradiation, these cells were pre-treated with PARPi (olaparib, 10 μM), ATM/ATRi (caffeine, 2 mM), ATMi (Ku55933, 5 μM), or ATRi (Ve-821, 10 μM), as indicated. The localization of GFP-Kif2C at the indicated time points is shown. The white lines mark the regions of laser micro-irradiation. Consistent results were observed in >10 cells for each treatment. ( E ) The DNA damage recruitment of Kif2C was examined as in panel D. The intensity of the GFP signal at laser-cut sites was normalized to that outside of the laser-cut sites for the relative enrichment of GFP-Kif2C. The mean values and standard deviations are shown (N = 5). ATM/ATRi showed similar kinetics as ATMi. P values were determined by two-tailed Student’s t-test (*<0.05, **<0.01, ***<0.001). ( F ) The N-terminus, middle segment ( M ), and C-terminus of MCAK was expressed with a GFP tag to examine their localization in laser-treated HeLa cells. The white lines mark the regions of laser micro-irradiation. Consistent results were observed in >10 cells for each segment. ( G ) The DNA damage recruitment of Kif2C-N, M, and C was examined as in panel F. The intensity of the GFP signal at laser-cut sites was normalized to that outside of the laser-cut sites for the relative enrichment. The mean values and standard deviations are shown (N = 5). ( H ) A series of truncation mutants were generated from the N-terminus of Kif2C. These mutants, tagged with GFP, were analyzed for recruitment to laser-stripes 10 min after the treatment. The result of positive or negative recruitment was determined by consistent results in >10 cells. ( I ) GFP-Kif2C deleted of aa 86–90 or neck-motif was expressed in HeLa cells which were micro-irradiated by laser (as marked by white lines). Both the aa 86–90 and neck-motif of Kif2C are required for the efficient recruitment of Kif2C to laser stripes. The white line marks the path of laser. ( J ) The recruitment to laser stripes, as in panel I, was quantified for Kif2C (WT, or deleted of the aa 86–90 or neck-motif). The intensity of the GFP signal at laser-cut sites was normalized to that outside of the laser-cut sites for the relative enrichment. The mean values and standard deviations are shown (N = 5, **p<0.01).

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Expressing, Irradiation, Immunofluorescence, Imaging, Extraction, Two Tailed Test, Generated

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: Two representative examples of Kif2C and 53BP1 foci were tracked in U2OS cells. The coordinated movements of WT-KIF2C and 53BP1 foci are shown.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques:

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) HeLa cells were treated with 10 Gy IR, the immunofluorescent (IF) signals of Kif2c and γ-H2AX are shown. Pre-extraction was performed by placing the dish on ice for 5 min with 0.1% Triton X-100 in 10 mM HEPES (pH 7.4), 2 mM MgCl2, 100 mM KCl, and 1 mM EDTA. ( B ) Kif2C and γ-H2AX IF was performed as in panel A, in Kif2C knockout (KO) cells. ( C ) The localization of GFP-Kif2C deleted of N-terminus before or after laser treatment is shown. The white line marks the path of laser. ( D ) Quantification of cells showing recruitment to laser stripes.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Extraction, Knock-Out

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: GFP-Kif2C (WT or deletion mutants) IP was performed using anti-GFP or control (blank) beads, as in . 10% input, control and GFP IP samples were analyzed by immunoblotting.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Control, Western Blot

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) HeLa cells were treated with Kif2C siRNA for 1 and 3 days, or with IR at 1 or 2 Gy (followed by 30 min incubation), as indicated. These cells were then harvested and analyzed by immunoblotting. ( B ) Kif2C gene deletion was carried out using the CRISPR-Cas9 technique in U2OS cells. Cell lysates were collected and analyzed by immunoblotting. ( C ) The comet assay was performed in control or Kif2C knockout (KO) U2OS cells, as described in Materials and methods. The percentage of DNA in the tail section was quantified, the mean values and standard derivations are shown (N > 20). Representative images are shown below. ( D ) HeLa cells were treated with control siRNA or Kif2C siRNA, and reconstituted with siRNA resistant GFP-Kif2C (WT or G495A), as indicated. Cell lysates were harvested and analyzed by immunoblotting. ( E ) Control or Kif2C knockout (KO) U2OS cells were transfected with WT or R420S Kif2C tagged with GFP, as indicated. One day after transfection, the samples were analyzed by immunoblotting. ( F ) U2OS Kif2C knockout (KO) cells were transfected with WT or Δ86–90 Kif2C tagged with GFP, as indicated. One day after transfection, the samples were analyzed by immunoblotting. ( G ) Asynchronized HeLa cells were treated with 20 µM DHTP for 3 hr, as indicated. The cell lysates were analyzed by immunoblotting. ( H ) HeLa cells were first synchronized at G1/S by thymidine-arrest, and then treated with 10 µM DHTP for 3 hr. The cell lysates were analyzed by immunoblotting. ( I ) HeLa cells were incubated in cisplatin (6.7 μM) and Kif2C siRNA, as indicated. The relative cell viability was determined by normalizing the cell number to that of the first day. The mean values and standard deviations, calculated from three independent experiments, are shown. *p<0.05. ( J ) HeLa cells were treated as in panel I for 2 days, and measured by the trypan blue exclusion assay for cell death. The mean values and standard deviations, calculated from three independent experiments, are shown. ( K ) WT or Δ86–90 Kif2C was expressed in Kif2C KO cells as in panel F. 1 day after transfection, these cells, along with control U2OS, were treated with various doses of etoposide for 2 days. The relative cell viability was determined by first calculating the ratio of cell number in day 3 to that in day 1, and then normalizing the ratio of etoposide treated cells to that of the untreated. The mean values and standard deviations, calculated from three independent experiments, are shown.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Incubation, Western Blot, CRISPR, Single Cell Gel Electrophoresis, Control, Knock-Out, Transfection, Trypan Blue Exclusion Assay

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: Representative foci formation is shown in Kif2C KO cells with or without expression of WT or G495A Kif2C, as in . Quantifications of foci number are shown on the right panels (N > 20).

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Expressing

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) The recruitment of GFP-Kif2C R420S and G495A to laser-induced DNA damage sites. The GFP localization before or after laser treatment is shown. The white line marks the path of laser. ( B ) WT or Kif2C KO U2OS cells were analyzed by FACS for cell cycle distribution. Cells were mock transfected or transfected with WT, G495A or R420S Kif2C, as in .

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Transfection

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) Kif2C depletion does not further induce DNA damage in cells treated with nocodazole (100 ng/ml). Control or Kif2C knockout (KO) U2OS cells were treated with nocodazole for 8 hr, as indicated. The samples were analyzed by immunoblotting. ( B ) Human head and neck cancer SCC38 cells were incubated in cisplatin (6.7 μM) and Kif2C siRNA, as indicated. The relative cell viability was determined by normalizing the cell number to that of the first day. The mean values and standard deviations, calculated from three independent experiments, are shown. ( C ) SCC38 cells were treated as in panel B for 2 days, and measured by the trypan blue exclusion assay for cell death. The mean values and standard deviations, calculated from three independent experiments, are shown. ( D ) HeLa cells were incubated in cisplatin (6.7 μM) and DHTP (10 μM), as indicated. The relative cell viability was determined by normalizing the cell number to that of the first day. The mean values and standard deviations, calculated from three independent experiments, are shown.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Control, Knock-Out, Western Blot, Incubation, Trypan Blue Exclusion Assay

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A, B, C ) HeLa cells treated with control ( A ) or Kif2C siRNA ( B ) were irradiated with 4 Gy IR, and incubated as indicated. The cell lysates were analyzed by immunoblotting. Quantification is shown in panel C. ( D, E, F ) U2OS cells, control ( D ) or Kif2C knockout (KO, ( E ), were irradiated with 4 Gy IR, and incubated as indicated. The cell lysates were analyzed by immunoblotting. Quantification is shown in panel F (as relative to 1 hr time point in control HeLa cells). ( G, H ) Chromosome-integrated, I-SceI-induced NHEJ ( G ) or HR ( H ) reporter systems are illustrated in the upper panels. These reporter cells were transfected with control or Kif2C siRNA. DNA repair was measured by immunoblotting of GFP expression in relative to β-actin expression. The GFP/β-actin ratio in Kif2C-depleted cells was normalized to that in control cells for relative repair efficiency. The mean values and standard deviations, calculated from three independent experiments, are shown. Statistical significance was analyzed using an unpaired 2-tailed Student’s t-test (***p<0.001). Kif2C depletion did not impact the expression of I-SceI .

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Control, Irradiation, Incubation, Western Blot, Knock-Out, Transfection, Expressing

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: Comet assay was performed in U2OS cells with or without Kif2C knockout. The cells were treated with doxorubicin at time 0 for 1 hr. The percentage of DNA in the tail section was quantified, the mean values and standard derivations are shown (N > 10).

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Single Cell Gel Electrophoresis, Knock-Out

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) Examples of 10 min mobility traces of EGFP-53BP1 foci in WT and Kif2c knockout (KO) U2OS cells after etoposide (20 μM) treatment. Kif2C depletion did not impact 53BP1 expression . ( B ) Mean-square displacement measurements of EGFP-53BP1 foci in WT and Kif2C KO U2OS cells, shown in black in red, respectively. ( C ) Mean-square displacement measurements of EGFP-53BP1 foci in WT U2OS cells treated with the vehicle control (DMSO), Taxol (5 μM), or DHTP (20 μM), as indicated. ( D ) Quantification of the distance travelled by EGFP-53BP1 foci over 10 min in the corresponding cells described in B-C. ( E ) Examples of disappearance (yellow arrowheads) and fusion (red circle) events of EGFP-53BP1 foci induced by etoposide in U2OS cells. ( F, G ) Number of EGFP-53BP1 foci in WT or Kif2C KO U2OS cells, treated with the vehicle control (DMSO), Taxol, or DHTP. These inhibitors were added either 5 min before ( F ) or 5 min after ( G ) etoposide treatment. ( H–J ) Numbers of fusion ( H ) and disappearance ( I–J ) events of EGFP-53BP1 foci in the corresponding cells in panel G are shown. A total of 15 randomly selected cells were analyzed over three independent experimental runs. For disappearance events, number of occurrence in the first 30 min under each treatment condition is shown in ( I ) and the time required for foci disappearance (min) over the entire hour of recording is shown in ( J ) (>150 events quantified per condition). The box represents 50% of the foci disappearance events and the line shows the median of the data set. All microscopy image acquisitions began five minutes after final compound treatment, either every 30 s for 10 min ( A–D ) or every 3 min for one hour ( H–J ). All data were collected from at least three independent experimental sets. Error bars, S.D.; ns: p>0.05; *p≤0.05; **p≤0.01; ***p≤0.001, by Student’s t -test.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Knock-Out, Expressing, Control, Microscopy

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A ) Representative examples of 10 min mobility traces of EGFP-53BP1, mCherry-CENP-B and EGFP-PRPF6 in U2OS cells. The tracked traces were overlaid over the corresponding images (upper panel). The lower panel shows the same traces aligned with a common starting point (indicated by circles). Note that the randomness of 53BP1 traces and the coordinated movement of the CENP-B and PRPF6 foci, representing general nuclear dynamics. ( B ) Quantification of the distance travelled by EGFP-53BP1, mCherry-CENP-B and EGFP-PRPF6 foci over 10 min in WT-U2OS and KIF2C-KO-U2OS cells. 53BP1 mobility was tracked 5 min after etoposide treatment, while the mobility of nuclear CENP-B and PRPF6 was tracked either with or without the addition of etoposide. ( C ) Examples of the intra-nuclear foci localization of CENP-B and PRPF6.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques:

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: Kif2C depletion increased g-H2AX induced by etoposide ( B ).

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques:

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: ( A–B ) Mean-square displacement measurements of EGFP-53BP1 foci in WT ( A ) and Kif2C KO ( B ) U2OS cells treated with the vehicle control (DMSO), the PARP1 inhibitor olaparib (10 μM), or the ATM inhibitor KU55933 (20 μM), as indicated. More than 50 foci were analyzed in three independent experiments. ( C ) Quantification of the distance travelled by EGFP-53BP1 foci over 10 min in the corresponding cells described in A-B. Experimental set up and image acquisition were the same as described in . ( D ) Kif2C mediates DSB end mobilization and the formation of DNA damage foci (model). Kif2C is recruited to DSB ends in a manner that depends on both ATM and PARP activities. Mediated by its MT depolymerase activity, Kif2C promotes the movement, and the subsequent clustering, of DSB ends. Therefore, Kif2C is an important downstream factor of the PARP and ATM-mediated DNA damage response that governs the mobility and dynamics of DSB ends.

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Control, Activity Assay

Journal: eLife

Article Title: Kinesin Kif2C in regulation of DNA double strand break dynamics and repair

doi: 10.7554/eLife.53402

Figure Lengend Snippet: As shown in , Mean-square displacement measurements of EGFP-53BP1 foci were measured in WT and Kif2C KO U2OS cells treated with the vehicle control (DMSO), the PARP1 inhibitor Olaparib (10 μM), or the ATM inhibitor KU55933 (20 μM). Quantification of the distance travelled by EGFP-53BP1 foci over 10 min in the corresponding cells, as in , is presented here in three groups (DMSO, Olaparib, and Ku55933).

Article Snippet: The human Kif2C expression vector was obtained from Addgene (mEmerald-MCAK-C-7, a gift from Michael Davidson via Addgene, plasmid # 54161).

Techniques: Control