Journal: Nucleic acids research

Article Title: Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells.

doi: 10.1093/nar/30.4.1009

Figure Lengend Snippet: Figure 1. Expression of HA-Rad51D in human cells and interaction of XRCC2 with HA-Rad51D. (A) Western blots with α-HA antibody of cell extracts from HeLaS3 subclones transfected with HA-Rad51D expression vector. Clones HD19 and HD25 expressed HA-Rad51D at a molecular weight at ∼36 kDa while clones HD16 and HD21 show little or no expression. (B) Immunoprecipitation of HA-Rad51D with HA affinity matrix in HD16 and HD19 extracts with or without irradiation. HA-Rad51D was pulled down in HD19 and detected on western blot with HA antibody. Native XRCC2 was specifically detected in the HA-Rad51D precipitates using either rabbit XRCC2 (α-XRCC2r) or mouse XRCC2 (α-XRCC2m) antibody. (C) HA-Rad51D was immunoprecipitated in HD19 extracts using HA affinity matrix at increasing concentrations of NaCl. XRCC2 was co-precipitated under all the conditions tested.

Article Snippet: The polyclonal antibodies against peptides of XRCC2, XRCC3, Rad51B and Rad51D were obtained from Novus Biologicals (Littleton, CO).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Clone Assay, Molecular Weight, Immunoprecipitation, Irradiation

Journal: Nucleic acids research

Article Title: Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells.

doi: 10.1093/nar/30.4.1009

Figure Lengend Snippet: Figure 2. Co-immunoprecipitation of Rad51D, Rad51C and XRCC2. (A) Immunoprecipitation of HD16 and HD19 extracts with α-XRCC2 (Novus, lane 1), α-Rad51D (Novus, lane 2) and α-HA (lanes 3 and 4). The western blot was probed with α-Rad51D. The native Rad51D and HA-Rad51D are indicated. The dark band at ∼55 kDa (lanes 1 and 2) is the heavy chain of rabbit IgG [the same in (B)]. (B) Immunoprecipitation of HD16 and HD19 extracts with α-XRCC2 (Novus, lane 1) and α-HA (lanes 2 and 3). The XRCC2 (top) and Rad51C (bottom) were detected using α-XRCC2r and α-Rad51C antibody, respectively. Rad51C co-precipitates with XRCC2 (lane 1) or HA-Rad51D (lane 3).

Article Snippet: The polyclonal antibodies against peptides of XRCC2, XRCC3, Rad51B and Rad51D were obtained from Novus Biologicals (Littleton, CO).

Techniques: Immunoprecipitation, Western Blot

Journal: Nucleic acids research

Article Title: Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells.

doi: 10.1093/nar/30.4.1009

Figure Lengend Snippet: Figure 3. Interaction of XRCC3 with Rad51C but not with Rad51D and XRCC2. The cells were incubated for 2 h after 8 Gy γ-irradiation. (A) Immunoprecipitation of control HeLa and H158 (expressing HA-XRCC3) cell extracts with HA affinity matrix. The western blots were incubated with α-HA (top) or α-HsRad51 (bottom). HA-XRCC3 is detected in H158 cells but not in the untransfected control, and Rad51 co-precipitates with HA-XRCC3. (B) Immunoprecipitation analysis in H158 and HD19 (expressing HA-Rad51D) was done using HA affinity matrix. The western blots were incubated with: a, α-Rad51D; b, α-XRCC3; c, α-Rad51C; and d, α-XRCC2r.

Article Snippet: The polyclonal antibodies against peptides of XRCC2, XRCC3, Rad51B and Rad51D were obtained from Novus Biologicals (Littleton, CO).

Techniques: Incubation, Irradiation, Immunoprecipitation, Control, Expressing, Western Blot

Journal: Nucleic acids research

Article Title: Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells.

doi: 10.1093/nar/30.4.1009

Figure Lengend Snippet: Figure 5. Lack of association of XRCC2 with Rad51. (A) Immunoprecipitation with HA affinity matrix in HD16 and HD19 cell extracts. The blots were incubated with α-HA (top) or α-HsRad51 (bottom). (B) Immunoprecipitation with Flag affinity matrix in wild type, irs1 and Flag-XRCC2 transformants of irs1 (IFX200). The blots were incubated with α-XRCC2r (top) or α-MmRad51 (bottom). (C) Immunofluorescence detection of GFP-XRCC2 in stable transformants of wild-type V79 cells 2 h after 0 or 8 Gy γ-irradiation (top). The same cells stained with DAPI (bottom).

Article Snippet: The polyclonal antibodies against peptides of XRCC2, XRCC3, Rad51B and Rad51D were obtained from Novus Biologicals (Littleton, CO).

Techniques: Immunoprecipitation, Incubation, Immunofluorescence, Irradiation, Staining

Journal: Nucleic acids research

Article Title: Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells.

doi: 10.1093/nar/30.4.1009

Figure Lengend Snippet: Figure 6. Speculative model of complexes and dynamic interactions among Rad51 paralogs and HsRad51. The diagram takes into account the results by Braybrooke and co-workers showing an XRCC2–Rad51D dimer in cell extracts (31) and the data from yeast two-hybrid analyses showing that Rad51B and Rad51C can form a dimer (30). XRCC2–Rad51D and Rad51B–Rad51C dimers combine to form the Rad51B–C–D–XRCC2 complex. XRCC3 may promote the dissociation of Rad51C from this complex to produce the XRCC3–Rad51C dimer that binds to Rad51. A possible conformational change as depicted for Rad51C could contribute to the specificity of interactions.

Article Snippet: The polyclonal antibodies against peptides of XRCC2, XRCC3, Rad51B and Rad51D were obtained from Novus Biologicals (Littleton, CO).

Techniques:

Journal: Cell reports

Article Title: XRCC2 Regulates Replication Fork Progression during dNTP Alterations.

doi: 10.1016/j.celrep.2018.11.085

Figure Lengend Snippet: Figure 1. XRCC2 Is Required for Efficient Fork Slowdown during Nucleotide Pool Depletion (A) Representative set of DNA fibers to display fork slowing induced by HU (500 mM) in U2OS cells treated with the indicated shRNAs. #1 and #2 indi- cate two independent shRNAs. (B) 5-Iodo-2’-deoxyuridine (IdU) to 5-chloro-2’- deoxyuridine (CldU) ratios of fibers from cells as shown in (A). (C) Quantification of fork restart in indicated cells after release from 2-hr HU treatment (2 mM). Ex- amples of various types of tracts are shown. Stalled and restarted replication forks are shown as per- centage of all CldU-labeled tracks. (D) IdU to CldU tract length ratios in U2OS cells after indicated knockdowns to study fork degradation following HU treatment (4 mM). A representative set of DNA fibers is shown. (E) Quantification of fork asymmetry in control and XRCC2-depleted U2OS cells following HU treat- ment (500 mM). Examples of symmetric and asym- metric sister forks are shown. (F) QIBC of cells exposed to indicated doses of HU for 45 min and immunostained for cyclin A and gH2AX. A.U., arbitrary units. DNA fiber labeling protocol is shown for individual panels. A minimum of 100 DNA fibers were analyzed for each condition. Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001. n.s., nonsignifi- cant. Wherever not explicitly indicated, shRNA#1 was used. See also Figure S1.

Article Snippet: Antibodies to the following proteins were used: a-tubulin (1:1000, sc-5286, Santa Cruz), RRM1 (1:200, sc-377426, Santa Cruz), HSP90 (1:500, sc-69703, Santa Cruz), RAD51 (1:50 for IF, sc-8349, Santa Cruz), XRCC2 (1:250, sc-365854, Santa Cruz), XRCC3 (1:250, sc-271714, Santa Cruz), RAD51B (1:250, sc-377192, Santa Cruz), RAD51D (1:100, sc-53432, Santa Cruz), PCNA (1:500, sc-56, Santa Cruz), H2AX (1:250, sc-54606, Santa Cruz), MCM3 (1:250, sc-365616, Santa Cruz) and MCM7 (1:250, sc-56324, Santa Cruz), RRM2 (1:1000, ab172476, Abcam), pKAP1 S824 (1:1500, ab70369, Abcam), pChk1 S345 (1:1500, ab58567, Abcam) pChk2 T68 (1:1500, ab85743, Abcam), HA-tag (1:1000, 10952100, Roche).

Techniques: Labeling, Control, shRNA

Journal: Cell reports

Article Title: XRCC2 Regulates Replication Fork Progression during dNTP Alterations.

doi: 10.1016/j.celrep.2018.11.085

Figure Lengend Snippet: Figure 2. XRCC2-Mediated Fork Slowing Is Specific to dNTP Alterations and Independent of ROS Signaling and Fork Reversal (A) Representative images for DNA fibers showing fork slowing in cells treated with genotoxic agents (NT, nontreated; 500 mM HU; 50 nM camptothecin [CPT]; 50 mM H2O2). (B) Quantification of IdU to CldU tract length ratios from cells as shown in (A). (C) IdU to CldU ratios in cells treated with aphidicolin (100 nM). (D) HU-induced fork slowing in cells pretreated with olaparib (10 mM). (E) Fork slowing in cells treated with indicated con- centrations of HU following pretreatment with NAC (5 mM). (F) Replication rate in cells treated with indicated concentrations of 5-FU for 48 hr. DNA fiber labeling protocol is shown for individual panels. A minimum of 100 DNA fibers were analyzed for each condition. Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001. n.s., nonsignificant. See also Figure S2.

Article Snippet: Antibodies to the following proteins were used: a-tubulin (1:1000, sc-5286, Santa Cruz), RRM1 (1:200, sc-377426, Santa Cruz), HSP90 (1:500, sc-69703, Santa Cruz), RAD51 (1:50 for IF, sc-8349, Santa Cruz), XRCC2 (1:250, sc-365854, Santa Cruz), XRCC3 (1:250, sc-271714, Santa Cruz), RAD51B (1:250, sc-377192, Santa Cruz), RAD51D (1:100, sc-53432, Santa Cruz), PCNA (1:500, sc-56, Santa Cruz), H2AX (1:250, sc-54606, Santa Cruz), MCM3 (1:250, sc-365616, Santa Cruz) and MCM7 (1:250, sc-56324, Santa Cruz), RRM2 (1:1000, ab172476, Abcam), pKAP1 S824 (1:1500, ab70369, Abcam), pChk1 S345 (1:1500, ab58567, Abcam) pChk2 T68 (1:1500, ab85743, Abcam), HA-tag (1:1000, 10952100, Roche).

Techniques: Labeling

Journal: Cell reports

Article Title: XRCC2 Regulates Replication Fork Progression during dNTP Alterations.

doi: 10.1016/j.celrep.2018.11.085

Figure Lengend Snippet: Figure 3. XRCC2 Modulates Cellular dNTP Pools through Regulation of RRM2 (A) Replication rate in U2OS cells treated with indi- cated concentrations of HU for 12 hr. (B) Cell-cycle profiles of cells treated with nocoda- zole and indicated concentrations of HU for 12 hr. (C) Quantification of fluorescence-activated cell sorting (FACS) profiles as shown in (B). Data are representative of 2 independent experiments involving triplicates. (D) Western blot showing steady-state levels of RRM1 and RRM2 in siControl and siXRCC2-treated cells. A nonspecific band serves as loading control. Asterisks indicate nonspecific bands. (E) Quantification of mean intensity of nuclear RRM2 in cells exposed to indicated concentrations of HU for 45 min. A.U., arbitrary units. (F) dATP concentrations (normalized to NT) after exposure to indicated doses of HU for 45 min (mean ± SEM; n = 4 biological replicates). DNA fiber labeling protocol is shown for individual panels. A minimum of 100 DNA fibers were analyzed for each condition. Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001. n.s., nonsignificant. See also Figures S3 and S4.

Article Snippet: Antibodies to the following proteins were used: a-tubulin (1:1000, sc-5286, Santa Cruz), RRM1 (1:200, sc-377426, Santa Cruz), HSP90 (1:500, sc-69703, Santa Cruz), RAD51 (1:50 for IF, sc-8349, Santa Cruz), XRCC2 (1:250, sc-365854, Santa Cruz), XRCC3 (1:250, sc-271714, Santa Cruz), RAD51B (1:250, sc-377192, Santa Cruz), RAD51D (1:100, sc-53432, Santa Cruz), PCNA (1:500, sc-56, Santa Cruz), H2AX (1:250, sc-54606, Santa Cruz), MCM3 (1:250, sc-365616, Santa Cruz) and MCM7 (1:250, sc-56324, Santa Cruz), RRM2 (1:1000, ab172476, Abcam), pKAP1 S824 (1:1500, ab70369, Abcam), pChk1 S345 (1:1500, ab58567, Abcam) pChk2 T68 (1:1500, ab85743, Abcam), HA-tag (1:1000, 10952100, Roche).

Techniques: FACS, Western Blot, Control, Labeling

Journal: Cell reports

Article Title: XRCC2 Regulates Replication Fork Progression during dNTP Alterations.

doi: 10.1016/j.celrep.2018.11.085

Figure Lengend Snippet: Figure 4. ATR-Mediated Phosphorylation of XRCC2 Is Essential to Restrain DNA Synthe- sis during dNTP Alterations (A) IdU to CldU ratios to display fork slowing induced by HU (500 mM) in U2OS cells treated with the indi- cated shRNAs. #1 and #2 indicate two independent shRNAs. (B) Top: schematic showing BC and DX2 sub- complexes of RAD51 paralogs. Bottom: western blot analysis of XRCC2, RAD51B, and RAD51D following subcellular fractionation of RAD51D- depleted U2OS cells. Nucleoporin (NPC) serves as loading control for the soluble nuclear fraction. (C) XRCC2 S247 phosphorylation in nuclear frac- tions from HeLa cells synchronized at the indicated stages of the cell cycle. (D) XRCC2 S247 phosphorylation in HeLa cells treated with the indicated concentrations of HU for 4 hr. (E) XRCC2 S247 phosphorylation in ataxia-telangi- ectasia and Rad3-related (ATR)-depleted HeLa cells treated with HU (2 mM; 4 hr). (F) Western blot showing depletion of endogenous XRCC2 and expression of shRNA-resistant hemag- glutinin (HA)-tagged XRCC2 variants in U2OS cells. (G) Complementation of HU-induced fork slowing in XRCC2-depleted U2OS cells by shRNA-resistant XRCC2 variants as shown in (F). (H) I-SceI-induced HR frequency (normalized to control cells) in U2OS-sister chromatid recombina- tion (SCR) 18 cells depleted of XRCC2 and stably expressing the indicated shRNA-resistant XRCC2 variants. Ratio of phosphorylated to total XRCC2 is normal- ized to control conditions and indicated below the respective lanes in all panels. DNA fiber labeling protocol is shown for individual panels. A minimum of 100 DNA fibers were analyzed for each condition. Student’s t test, *p < 0.05; **p < 0.01; ***p < 0.001. n.s., nonsignificant. See also Figures S5 and S6.

Article Snippet: Antibodies to the following proteins were used: a-tubulin (1:1000, sc-5286, Santa Cruz), RRM1 (1:200, sc-377426, Santa Cruz), HSP90 (1:500, sc-69703, Santa Cruz), RAD51 (1:50 for IF, sc-8349, Santa Cruz), XRCC2 (1:250, sc-365854, Santa Cruz), XRCC3 (1:250, sc-271714, Santa Cruz), RAD51B (1:250, sc-377192, Santa Cruz), RAD51D (1:100, sc-53432, Santa Cruz), PCNA (1:500, sc-56, Santa Cruz), H2AX (1:250, sc-54606, Santa Cruz), MCM3 (1:250, sc-365616, Santa Cruz) and MCM7 (1:250, sc-56324, Santa Cruz), RRM2 (1:1000, ab172476, Abcam), pKAP1 S824 (1:1500, ab70369, Abcam), pChk1 S345 (1:1500, ab58567, Abcam) pChk2 T68 (1:1500, ab85743, Abcam), HA-tag (1:1000, 10952100, Roche).

Techniques: Phospho-proteomics, Western Blot, Fractionation, Control, Expressing, shRNA, Stable Transfection, Labeling

Journal: Cell reports

Article Title: XRCC2 Regulates Replication Fork Progression during dNTP Alterations.

doi: 10.1016/j.celrep.2018.11.085

Figure Lengend Snippet: Figure 5. Model for Regulation of Fork Progression by XRCC2-RAD51D during dNTP Alterations See Discussion section for details.

Article Snippet: Antibodies to the following proteins were used: a-tubulin (1:1000, sc-5286, Santa Cruz), RRM1 (1:200, sc-377426, Santa Cruz), HSP90 (1:500, sc-69703, Santa Cruz), RAD51 (1:50 for IF, sc-8349, Santa Cruz), XRCC2 (1:250, sc-365854, Santa Cruz), XRCC3 (1:250, sc-271714, Santa Cruz), RAD51B (1:250, sc-377192, Santa Cruz), RAD51D (1:100, sc-53432, Santa Cruz), PCNA (1:500, sc-56, Santa Cruz), H2AX (1:250, sc-54606, Santa Cruz), MCM3 (1:250, sc-365616, Santa Cruz) and MCM7 (1:250, sc-56324, Santa Cruz), RRM2 (1:1000, ab172476, Abcam), pKAP1 S824 (1:1500, ab70369, Abcam), pChk1 S345 (1:1500, ab58567, Abcam) pChk2 T68 (1:1500, ab85743, Abcam), HA-tag (1:1000, 10952100, Roche).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: XRCC2 protein was expressed highly in T84 colon cancer cell line. Total protein of various tumor cell lines (colon cancer cell lines T84, HT29 and Lovo, breast cancer cell line MCF-7, esophageal cancer cell line EC9706) and normal HEK293 cell line were extracted, and the XRCC2 expression was examined by Western blot.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: XRCC2 expression was suppressed by vector-based shRNA in T84 cells. ( A ) XRCC2 protein expression; and ( B ) XRCC2 mRNA expression. Cells were transfected with either shRNA-XRCC2 or shRNA-SC. Total protein and mRNA levels of XRCC2 were determined at 24 h after transfection by Western blot and quantitative real-time polymerase chain reaction (PCR) analyses, respectively. The values are presented as the mean ± SD ( n = 6). ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Expressing, Plasmid Preparation, shRNA, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Knockdown of XRCC2 by shRNA inhibited cell growth of T84 cells. Cells were transfected with either shRNA-XRCC2 or shRNA-SC. The effect of XRCC2 suppression on cell growth in T84 cell line was examined by MTT assay. The values are presented as the mean ± SD ( n = 12). * p < 0.05, ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, shRNA, Transfection, MTT Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Knockdown of XRCC2 by shRNA enhanced radiosensitivity to X-radiation of T84 cells. ( A ) Photographs of colonies; and ( B ) the number of colonies. Cells were treated with either shRNA-XRCC2, shRNA-SC, radiation or shRNA-XRCC2 combined radiation. Cells of control group, shRNA-SC group and shRNA-XRCC2 group were not exposed to radiation. Cells of radiation group were treated with 8 Gy X-ray radiation after T84 cells attached. shRNA-XRCC2 combined radiation group received 8 Gy radiation at 12 h after shRNA-XRCC2 transfection. The effect of XRCC2 suppression on radiosensitivity of T84 cell line was examined by colony formation assay. The values are presented as the mean ± SD ( n = 6). * p < 0.05, ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, shRNA, Control, Transfection, Colony Assay

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Knockdown of XRCC2 arrested cells in the G2/M phase. ( A ) Cell cycle distribution without exposure to radiation; and ( B ) cell cycle distribution after exposure to 8 Gy radiation. Cells were transfected with either shRNA-XRCC2 or shRNA-SC. Cells were irradiated with 8 Gy at 12 h after transfection. The effect of XRCC2 suppression on cell cycle distribution in T84 cell line was examined by flow cytometric analysis. The values are presented as the mean ± SD ( n = 5). * p < 0.05, ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, Transfection, shRNA, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Knockdown of XRCC2 enhanced cell apoptosis after radiation treatment. ( A ) Cell apoptosis without exposure to radiation; and ( B ) cell apoptosis after exposure to 8 Gy radiation. Cells were transfected with either shRNA-XRCC2 or shRNA-SC. Cells were irradiated with 8 Gy at 12 h after transfection. The effect of XRCC2 suppression on cell apoptosis in T84 cell line was examined by flow cytometric analysis. The values are presented as the mean ± SD ( n = 5). ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, Transfection, shRNA, Irradiation, Control

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Knockdown of XRCC2 decreased tumor growth in nude mice. ( A ) Tumor growth curve; ( B ) Tumor weight after mice were sacrificed; ( C ) Ectopic T84 xenografts in nude mice; and ( D ) Photograph of the dissected tumors. The effect of shRNA-XRCC2 on tumorigenicity was examined in nude mice. Mice were treated with either shRNA-XRCC2 or shRNA-SC. Radiation treatment (2 Gy) was given once every other day when tumor volume reached 100 mm 3 in a total of 10 days. The tumor volume and tumor weight was examined for 28 days after exposure to radiation. The values are presented as the mean ± SD ( n = 10). ** p < 0.01 compared with the control group.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, shRNA, Control

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Pathological analysis of knockdown of XRCC2 expression. The effect of XRCC2 shRNA on pathology was examined in nude mice. Mice were treated with either shRNA-XRCC2 or shRNA-SC. Radiation treatment (2 Gy) was given once every other day when tumor volume reached 100 mm 3 in a total of 10 days. The tumor pathology was examined on day 28 after exposure to radiation. (Magnification: ×400; Scale bar: 100 μm).

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: Knockdown, Expressing, shRNA

Journal: International Journal of Molecular Sciences

Article Title: shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

doi: 10.3390/ijms15022157

Figure Lengend Snippet: Pathological analysis of harvested tumors in nude mice.

Article Snippet: T84 cells were stably transfected with either shRNA XRCC2 plasmid (sc-36861-SH, Santa Cruz, Dallas, TX, USA) (shRNA-XRCC2) or control shRNA plasmid-A (sc-108060, Santa Cruz) as scramble shRNA (shRNA-SC), using Lipofectin reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction.

Techniques: shRNA

Journal: DNA repair

Article Title: RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination

doi: 10.1016/j.dnarep.2019.02.008

Figure Lengend Snippet: A) Yeast 2-hybrid analysis showing the interactions of each of the RAD51 paralogs (and SWS1, a SWIM-domain containing protein) with each other. The PJ694a yeast strain was transformed with a plasmid where RAD51, the RAD51 paralogs (RAD51B, RAD51C RAD51D, SWSAP1, XRCC2, and XRCC3) and SWS1 were cloned into a plasmid that expresses the GAL4 DNA activating (pGAD) and GAL4 DNA binding (pGBD) domains.A yeast-2-hybrid interaction was assayed by platting the yeast on SC-Leu¯Trp¯His¯ and compared to the empty pGAD or pGBD plasmids, which were used as negative controls. Note that XRCC3 analysis had to be performed on a separate plate because of the plate size and that, consistent with the yeast Rad51 paralogs, human RAD51 Y2H interactions with the RAD51 paralogs are only observed when RAD51 is expressed in the pGAD vector [57].

Article Snippet: Antibodies against XRCC2 (TA327180, OriGene) was used at a 1:2,000 dilution.

Techniques: Transformation Assay, Plasmid Preparation, Clone Assay, Binding Assay

Journal: DNA repair

Article Title: RAD51D splice variants and cancer-associated mutations reveal XRCC2 interaction to be critical for homologous recombination

doi: 10.1016/j.dnarep.2019.02.008

Figure Lengend Snippet: A) Yeast 2-hybrid analysis of potential post-translationally modified residues in RAD51D on its interaction with XRCC2. The PJ694a yeast strain was transformed with a plasmid where RAD51D or the indicated RAD51D mutation was fused to the GAL4-DNA binding domain [BD; pGBD-RAD51D (WT)] and a plasmid where XRCC2 was fused to the GAL4-DNA activating domain (AD; pGAD-XRCC2). A yeast-2-hybrid interaction was assayed by platting the yeast on SC-Leu¯Trp¯His¯ (least stringent), SC-Leu¯Trp¯His¯+3AT (more stringent), or SC-Leu¯Trp¯His¯Ade¯(most stringent) and compared to the loading control (SC-Leu¯Trp¯). Empty AD (pGAD; Empty) plasmid was used as a negative control.

Article Snippet: Antibodies against XRCC2 (TA327180, OriGene) was used at a 1:2,000 dilution.

Techniques: Modification, Transformation Assay, Plasmid Preparation, Mutagenesis, Binding Assay, Negative Control