Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Survival curves of RECQL4 mRNA expression in all breast cancers from the database (Affymetrix ID for RECQL4 : 213520_at). Notes: ( A ) OS curve; ( B ) DMFS curve; ( C ) RFS curve; ( D ) PPS curve. Abbreviations: BC, breast cancer; OS, overall survival; DMFS, distant metastasis-free survival; RFS, relapse-free survival; PPS, postprogression survival.

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: OS curves of RECQL4 with the different intrinsic breast cancer subtypes identified for patients in the database (Affymetrix IDs for RECQL4 : 213520_at). Notes: ( A ) Luminal A; ( B ) HER2; ( C ) Luminal B; ( D ) Basal-like. Abbreviation: OS, overall survival.

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques:

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Correlation of RecQ-member mRNA expression with OS in different ER statuses of breast cancer patients

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Correlation of RecQ-member mRNA expression with OS in different PR statuses of breast cancer patients

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Correlation of RecQ-member mRNA expression with OS in different lymph-node statuses of breast cancer patients

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Correlation of RecQ-member mRNA expression with OS in different grades of breast cancer patients

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Correlation of RecQ-member mRNA expression with OS in different TP53 statuses of breast cancer patients

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing, Mutagenesis

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Prognostic significances of WRN and RECQL4 protein expression in breast cancer. Notes: ( A ) Immunohistochemical analysis of WRN in breast cancer tissue with low staining and high staining; ( B ) immunochemical analysis of RECQL4 in breast cancer tissue with low staining and high staining; ( C ) Kaplan–Meier OS analysis of WRN protein expression for patients with breast cancer; ( D ) Kaplan–Meier OS analysis of RECQL4 protein expression for patients with breast cancer. Each tissue section was observed under microscopy with low magnification of 50× and high magnification of 100×. Abbreviation: OS, overall survival.

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques: Expressing, Immunohistochemical staining, Staining, Microscopy

Journal: Cancer Management and Research

Article Title: Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members – RECQL , BLM , WRN , RECQL4 , and RECQL5 – in patients with breast cancer

doi: 10.2147/CMAR.S185769

Figure Lengend Snippet: Clinicopathological characteristics of WRN- and RECQL4-expression cohorts in tumor tissue

Article Snippet: IHC staining of the TMAs was performed using primary antibodies against WRN (ab200; Abcam, Cambridge, UK) or RECQL4 (25470002; Novus Biologicals, Littleton, CO, US).

Techniques:

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: RecQL4 expression analyses at the mRNA and protein levels in normal and prostate cancer cell lines of human origin. (A) RT-PCR analysis of RecQL4 and androgen receptor (AR) expression in normal immortalized human prostate epithelial cells (RWPE1) and four metastatic prostate cancer cell lines. GAPDH was used as a loading control. Quantitative real-time PCR analysis of RecQL4 expression in primary (PrEC), SV-40 immortalized (RWPE1) and metastatic prostate cancer cell lines (DU145, LNCaP and PC3). RecQL4 expression in normal and cancer cell lines was normalized to β-Actin. Bars indicate SEM. (B) Western blot analysis of RecQL4 expression in normal and prostate cancer cell lines. α-Tubulin was used as a loading control. (C) RT-PCR analysis of RecQL4 expression in non-tumorigenic (RWPE1), tumorigenic (RWPE2) and prostate cancer cell lines (ALVA31 and ALVA41). (D) Quantitative real-time PCR analysis of RecQL4 in non-tumorigenic (RWPE1) and tumorigenic (RWPE2) cells. Error bars indicate SEM.

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: (A) Immunohistochemical analysis of RecQL4 expression in normal (I), malignant (II & III) and metastatic prostate tumor tissues (IV–VI). Prostate tissue panel arrays (PR951 and PR751) purchased from US Biomax Inc., MD, USA were utilized. Malignant tumor samples shown in II (67 year old, Gleason score 7, PSA 37.3 ng/ml) and III (63 year old, Gleason score 10, PSA not determined) illustrate that the RecQL4 expression increases with increasing tumor grade. Samples: IV (61 year old male; metastatic adenocarcinoma to bone), V and VI (65 year old, metastatic adenocarcinoma to bone and abdominal wall respectively). (B) Staining pattern of a PIN sample (50 year old, Gleason grade III) is shown in the lower left bottom panel. Arrows indicate the regions of positive RecQL4 staining. (C) The cumulative average value of integrated optical density obtained for all the normal, malignant and metastatic samples are given. Bars indicate SEM. Scale bar = 50μM.

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: Summary of immunological staining analysis of RecQL4 in prostate tumor tissues

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: Staining

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: RecQL4 suppression leads to proliferation failure and apoptosis in metastatic prostate cancer cells. (A & B) Analysis of RecQL4 expression in RWPE1 and prostate cancer cell lines 72 hr after transfection with control and RecQL4 specific siRNA. (C) Effect of RecQL4 silencing on proliferation by CyQuant assay 48 hr after transfection with indicated concentrations of scrambled and RecQL4 specific siRNA in RWPE1 and PC3 cell lines. Error bars indicate SD. (D) Cell cycle analysis of immortalized RWPE1 and three metastatic prostate cancer cell lines (DU145, LNCaP and PC3) transfected with 25–100 nM of either scrambled control or RecQL4 specific siRNA. Cell cycle analysis was performed 72 hr after siRNA transfection.

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: Expressing, Transfection, CyQUANT Assay, Cell Cycle Assay

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: RecQL4 suppression leads to PARP-1 mediated apoptotic death in prostate cancer cells. (A) Analysis of cleaved PARP-1, Bax and AIF proteins in RWPE1 and PC3 cells after 72 hr of transfection with control and RecQL4 siRNA. (B) PC3 cells transfected with RecQL4 shRNA targeting vectors (TI339521 and TI339524) showed reduced survival. (C) Analysis of RecQL4 expression in empty vector (clone 5), scrambled vector (clone7) and RecQL4 shRNA (clones 6 and 4) transfected clonal cell lines of PC3.β-Actin was used as a loading control. (D) RecQL4 suppressed clonal cell lines (C6 and C4) showed increased focalization of PARP-1 and 53BP1. Western blot analysis showed increased γ-H2AX level in RecQL4 suppressed cells. Scale bar =10μM.

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: Transfection, shRNA, Expressing, Plasmid Preparation, Clone Assay, Western Blot

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: RecQL4 suppression drastically reduces cell invasiveness in vitro and tumorigenic growth in vivo. (A) RecQL4 suppressed clonal cell lines (C4 and C6) showed reduced cell invasion capacity. (B&C) Suppression of RecQL4 expression in prostate cancer cells reduces tumorigenicity in nude mice. Images of tumors dissected out from the sacrificed mice are shown in B. The tumor size (mm3) versus days of post-injection is shown in C. Difference in relative tumor volume observed at 4 weeks between empty vector and RecQL4 ShRNA transfected cells were found to be statistically significant. *p < 0.01.

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: In Vitro, In Vivo, Expressing, Injection, Plasmid Preparation, shRNA, Transfection

Journal:

Article Title: Human RecQL4 helicase plays critical roles in prostate carcinogenesis

doi: 10.1158/0008-5472.CAN-10-1743

Figure Lengend Snippet: Expression of RecQL4 expression is modulated by Rb-E2F1 pathway in prostate cancer cells. (A) Prostate cancer cells showed an inverse correlation between p16 level and Rb hyperphosphorylation (Ser807/811). TSA treatment abolished Rb hyperphosphorylation and reduced the expression of RecQL4 in both PC3 and RWPE1 cells. TSA treatment also reduced the levels of Cyclin A, Cyclin E and Cdk2 in PC3 cells but not in RWPE1 cells. GAPDH was used as a loading control. (B) TSA reduced the levels of E2F1 and phosphorylated Rb (T821) proteins in metastatic prostate cancer cell lines. (C) Quantitative real time PCR analysis showing the reduced expression levels of both E2F1 and RecQL4 in DU145 after TSA treatment. (D) Nucleotide sequence of RecQL4 promoter highlighting the putative responsive element for E2F1 binding is shown. The ChIP assay was performed in HeLa cells using antibodies specific for Rb and E2F1. Rb and E2F1 associated RecQL4 promoter DNA was amplified by PCR using appropriate primers. RNA polymerase II antibody was used as a positive control. Enrichment of the RecQL4 promoter in the immunoprecipitated DNA was quantified by real time PCR using the primers specific for RecQL4 promoter. Expon- Exponential curve.

Article Snippet: Empty (TR20002), scrambled (TR30003) and 4 different RecQL4 specific shRNA vectors (TI339521, TI339522, TI339523 and TI339524) were procured from Origene, USA (Cat. No#TR309882).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Sequencing, Binding Assay, Amplification, Positive Control, Immunoprecipitation

Journal: PloS one

Article Title: RecQL4 helicase amplification is involved in human breast tumorigenesis.

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: Figure 1. Amplification of RecQL4 genomic locus analyzed by mBAND-FISH and quantitative real time PCR. (A) Analysis of chromosome specific mBAND-FISH in the metaphase spreads of normal primary (HMEC), immortalized (MCF-10F) and tumorigenic breast cancer cell lines. Chromosomes and chromosome regions positive for the probe are shown in the inserts. (B) FISH analysis using spectrum orange labeled BAC (Bacterial Artificial Chromosome) probe proximal to 8q24.3 chromosome locus harboring the RecQL4 gene in MDA-MB453 and MDA-MB361 cells. The BAC probe was purchased from Open Biosystems (RP11–374B7, Huntsville, Alabama, USA). (C) Upper panel: Agarose gel electrophoresis showing the abundance of RecQL4 genomic DNA detected by PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used as an internal control; Lower panel: Abundance of RecQL4 genomic DNA detected by quantitative real time PCR in breast cancer cell lines relative to normal primary and immortalized breast epithelial cells. GAPDH was used for normalizing the values of RecQL4. doi:10.1371/journal.pone.0069600.g001

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Labeling, Agarose Gel Electrophoresis, Control

Journal: PloS one

Article Title: RecQL4 helicase amplification is involved in human breast tumorigenesis.

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: Figure 2. RecQL4 protein or mRNA level in breast tumor cell lines and clinical breast cancer specimens. (A) Western blot detection of RecQL4 protein level in HMEC, MCF-10F and five breast tumor cell lines. b-Actin was used to verify equal loading of proteins. (B) Analysis of RecQL4 expression by quantitative real time PCR in normal breast tissues and breast cancer specimens with different pathological grades. TissueScan breast cancer tissue qPCR array was purchased from Origene. RecQL4 expression detected in normal breast tissues was considered as 1. The data represent mean 6 SD from three independent experiments. doi:10.1371/journal.pone.0069600.g002

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: PloS one

Article Title: RecQL4 helicase amplification is involved in human breast tumorigenesis.

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: Figure 3. In vitro clonogenic survival and in vivo tumorigenic assays in MDA-MB453 tumor cells after knock-down of RecQL4 expression. (A) Western blot analysis of RecQL4 expression in parental, control shRNA (ShControl) and RecQL4 specific shRNA transduced MDA- MB453 cells (ShRecQL4-C5 & C8). RecQL4 expression was highly reduced in ShRecQL4-C5 and C8 stably selected by puromycin antibiotics. (B) Clonogenic survival assay was performed on ShRecQL4-C5 and C8 cells relative to parental and ShControl cells. Data presented at each time point were the mean value of eight cultures from two independent experiments. Bars indicate mean6SD. (C) Analysis of in vivo tumorigenicity of MDA- MB453 cells after RecQL4 silencing. Parental, ShControl and ShRecQL4-C5 & C8 cells were subcutaneously injected into seven immunosuppressed nude mice and tumor growth was monitored for 4 weeks. Tumor growth as a function of time is shown in the left panel. Images of tumors resected from mice are shown in the right panel. Note that 4 of 7 mice injected with either ShRecQL4-C5 or C8 cells did not show any tumor growth. Bars indicate mean6SD. doi:10.1371/journal.pone.0069600.g003

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: In Vitro, In Vivo, Knockdown, Expressing, Western Blot, Control, shRNA, Stable Transfection, Clonogenic Cell Survival Assay, Injection

Journal: PloS one

Article Title: RecQL4 helicase amplification is involved in human breast tumorigenesis.

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: Figure 4. RecQL4 associates with survivin. (A) Endogenous survivin was immunoprecipitated with Flag-RecQL4 recognized by an anti-Flag antibody from cell extracts of 106 U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were detected with antibody against survivin (Cell Signal). Five percent of the lysate was used for the loading control (Input) and the remaining 95% for co-immunoprecipitation. (B) Endogeneous RecQL4 was immunoprecipitated with Flag-survivin recognized by an anti-Flag antibody from cell extracts of 106 U2OS cells, but not with Flag-GFP. The immunoprecipitated proteins were visualized by Western blot analysis with antibody against RecQL4 (Cell signal). (C) In the upper panel, schematic diagram of RecQL4 deletion constructs used for Co-IP studies is shown. In the lower panel, 293T cells were co-transfected with pRc-CMV2- survivin and one of the Flag-tagged truncated RecQL4 expressing vectors: pFlag-RecQL4-NT(2), pFlag-RecQL4-HD(2) or pFlag-RecQL4-CT(2). Survivin was immunoprecipitated with N-terminal (NT) deleted Flag-RecQL4 protein, not with helicase domain (HD) or C-terminal (CT) deleted Flag- RecQL4 protein. doi:10.1371/journal.pone.0069600.g004

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Control, Western Blot, Construct, Co-Immunoprecipitation Assay, Transfection, Expressing

Journal: PloS one

Article Title: RecQL4 helicase amplification is involved in human breast tumorigenesis.

doi: 10.1371/journal.pone.0069600

Figure Lengend Snippet: Figure 5. RecQL4 modulates survivin expression. (A)Western blot analysis showed a reduced level of survivin expression and phospho-histone 3 (p-H3) in RecQL4 suppressed cells (ShRecQL4-C5 and C8) at 1 and 1.5 h post 0.25 mM H2O2 treatment. Bax protein did not show any alteration after oxidative DNA damage. b-Actin was used to verify equal loading of proteins. (B) Forced expression of RecQL4 in ShRecQL4-C5 and C8 cells led to an increased level of survivin expression at 48 h post transfection with pFlag-RecQL4 plasmid (lane 1) or plasmids (lane 2 & 3) containing nucleotide substitution at RecQL4 ShRNA-targeted area without changing the amino acid sequence. doi:10.1371/journal.pone.0069600.g005

Article Snippet: Both RecQL4 shRNA and scrambled control shRNA were procured from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, shRNA, Sequencing

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: Ionizing radiation stimulates the interaction between RECQL4 and DNA-PK cs . ( A ) Identification of RECQL4-associated proteins following DNA damage. U2OS cells were irradiated with 10 Gy of IR, allowed to recover for 10 min, and RECQL4 was immunoprecipiated from whole cell lysates. The samples were resolved via SDS-PAGE, stained, and then analyzed using mass spectrometry analysis. Shown are the top 17 RECQL4-interacting proteins identified in the screen. DNA-PK cs (gene: PRKDC ) was a top hit in the RECQL4 protein-protein interaction screen. ( B ) DNA damage promotes the interaction between RECQL4 and the DNA-PK complex (Ku70/80 heterodimer and DNA-PK cs ). U2OS cells were mock treated or irradiated with 10 Gy and allowed to recover for 10 min. Endogenous RECQL4 was immunoprecipitated and its interaction with the DNA-PK complex was assessed via immunoblotting using DNA-PK cs , Ku70 and Ku80 antibodies. ( C ) DNA-PK cs was immunoprecipitated from HCT116 wild-type (WT) or DNA-PK cs knockout (DNA-PKcs KO) cells that were treated with 10 Gy of IR and allowed to recover for 10 min. The samples were resolved via SDS-PAGE and the interaction between DNA-PK cs and RECQL4 and Ku70/80 was determined via immunoblotting. ( D ) DNA-PK cs was immunoprecipitated from U2OS wild-type (WT) or RECQL4 knockout (RQ4KO) cells that were irradiated with 10 Gy of IR and allowed to recover for 10 min. The samples were resolved via SDS-PAGE and interaction between RECQL4 and DNA-PK cs and Ku70/80 was determined via immunoblotting.

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Irradiation, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Knock-Out

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: RECQL4 promotes DNA-PK-mediated DNA end bridging. ( A, B ) Initial recruitment of YFP-tagged Ku80 ( A ) and DNA-PK cs ( B ) to laser-induced DSB is not affected by knockdown of RECQL4 in U2OS cells. Relative fluorescent intensity of YFP-tagged Ku80 and DNA-PK cs in U2OS cells treated with RECQL4 siRNAs (RQ4KD) or control siRNAs (Ctrl) following micro-irradiation are presented as mean ± standard error of the mean (SEM). The knockdown efficiency is shown in . Samples analyzed were 11 Ctrl and 13 RQ4KD cells for YFP-tagged Ku80 and 13 Ctrl and 19 RQ4KD cells for YFP-tagged DNA-PK cs . Student's t-test (two-sided) was performed to assess statistical significance (n.s. = not significant). ( C, D ) Depletion of RECQL4 attenuates the accumulation of Ku80 ( C ) and DNA-PKcs ( D ) at laser-induced DSBs. Relative fluorescent intensity of GFP-tagged Ku80 and DNA-PK cs following micro-irradiation are presented as mean ± SEM. The knockdown efficiency is shown in . Samples analyzed were 19 Ctrl siRNA treated cells and 17 RQ4KD cells for YFP-tagged Ku80, and 12 Ctrl and 18 RQ4KD cells for YFP-tagged DNA-PK cs . Student's t-test (two-sided) was performed to assess statistical significance (* P < 0.05 and ** P < 0.01). ( E ) RECQL4 stimulates end bridging of dsDNA end by DNA-PK in vitro . The indicated purified proteins were incubated with a biotin-labeled 36 bp dsDNA with one end linked to streptavidin beads and a 50 bp dsDNA with radioactive 32 P labeled, the DNA-protein complex were pulled down, washed, and dotted on a nylon membrane for radiography. The signal indicates amount of free dsDNA (50bp) bridged with beads-bound dsDNA (36bp) by proteins. The data were generated from three independent experiments and presented as mean ± SEM. Two-sided student's t-test (two-sided) was performed to assess statistical significance (* P < 0.05).

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Knockdown, Control, Irradiation, In Vitro, Purification, Incubation, Labeling, Membrane, Generated

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: RECQL4 promotes stabilization of NHEJ machinery at DSBs. ( A, B ) NHEJ protein–protein interactions are significantly decreased in the absence of RECQL4. DNA-PK cs ( A ) and Ku70 ( B ) were immunoprecipitated from U2OS wild-type (WT) or RECQL4 knockout (RQ4KO) cells that were treated with 10 Gy of IR and allowed to recover for 10 minutes. The samples were resolved via SDS-PAGE and interactions between DNA-PK cs and Ku70 with NHEJ factors (LIG4, XRCC4, XLF and/or Artemis) and RECQL4 were determined via immunoblotting. ( C , D ) RECQL4 promotes recruitment of XRCC4 and XLF to laser-generated DSBs. Relative fluorescent intensity of GFP-tagged XRCC4 ( C ) and XLF ( D ) in WT or RQ4KO cells following micro-irradiation are presented as mean ± standard error of the mean (SEM). For XRCC4, the results were calculated from 7 WT and 12 KO cells and 9 WT and 10 RQ4KO cells for XLF. Student's t -test (two-sided) was performed to assess statistical significance (* P < 0.05, ** P < 0.01 and *** P < 0.001). ( E ) Recruitment of NHEJ core factors to chromatin after IR is attenuated in RECQL4 knockout (RQ4KO) cells. U2OS wild-type (WT) and RQ4KO cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Subsequently, cytoplasmic, soluble nuclear, and chromatin fractions were isolated for immunoblotting to examine the recruitment of proteins listed in the figure to the chromatin after irradiation.

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Protein-Protein interactions, Immunoprecipitation, Knock-Out, SDS Page, Western Blot, Generated, Irradiation, Isolation

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: The kinase activity of DNA-PK cs promotes the accumulation of RECQL4 at DSBs. ( A ) Inhibition of DNA-PK cs attenuates accumulation of RECQL4 at laser-induced DSBs. U2OS RECQL4 Knockout cells stably expressing GFP-tagged RECQL4 were pretreated either with DMSO or 10 μM NU7441 for 2 h, and subsequently laser micro-irradiation assays were performed. Relative fluorescent intensity of GFP-tagged RECQL4 following micro-irradiation are presented as mean ± standard error of the mean (SEM). Samples analyzed were 15 for DMSO-treated and 16 NU7441-treated cells. Student's t -test (two-sided) was performed to assess statistical significance (*** P < 0.001). ( B ) Inhibition of DNA-PK cs reduces the recruitment of RECQL4 to the chromatin fraction following IR. U2OS cells were pretreated with 10 μM NU7441 or DMSO for 2 h, mock-treated or irradiated with a dose of 10 Gy, and allowed to recover for 10 min. Subsequently, cytoplasmic, soluble nuclear and chromatin fractions were isolated for immunoblotting to examine the recruitment of proteins listed in the figure to the chromatin after irradiation. ( C ) Accumulation of GFP-tagged RECQL4 in HCT116 DNA-PK cs kinase-dead (KD/–) cells is reduced compared to that in control DNA-PK cs +/– cells (+/–). Relative fluorescent intensity of GFP-tagged RECQL4 following micro-irradiation are presented as mean ± standard error of the mean (SEM). Samples analyzed were 8 for +/– and 8 for KD/– cells. Student's t-test (two-sided) was performed to assess statistical significance (*** P < 0.001). (D) Recruitment of RECQL4 to the chromatin fraction is decreased in KD/– cells compared to +/– cells. KD/– and +/– cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 minutes. Subsequently, cytoplasmic, soluble nuclear and chromatin fractions were isolated for immunoblotting to examine the recruitment of proteins listed in the figure to the chromatin after irradiation.

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Inhibition, Knock-Out, Stable Transfection, Expressing, Irradiation, Isolation, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: RECQL4 is phosphorylated by DNA-PK. ( A ) RECQL4 is phosphorylated at the S/T-Q motif after treatment with ionizing radiation (IR). HEK293T cells transiently expressing 3XFLAG-tagged RECQL4 were mock treated or irradiated with 10 Gy and allowed to recover for 20 min. The cells were lysed under denaturing conditions and 3XFLAG-tagged RECQL4 was purified using FLAG-M2 beads and subsequently mock treated or treated with lambda phosphatase (λPP). The samples were resolved via SDS-PAGE and immunoblotting was performed using a phospho-S/T-Q motif antibody. ( B ) IR-induced phosphorylation of RECQL4 at the S/T-Q motif is time dependent. HEK293T cells expressing 3XFLAG-tagged RECQL4 were irradiated with 10Gy and lysed at the time points indicated in the figure. Samples were processed and analyzed as stated in (A). ( C ) IR-induced phosphorylation of RECQL4 at the S/T-Q motif is increased in G1 phase of the cell cycle. RECQL4 knockout U2OS cells expressing 3XFLAG-RECQL4 were synchronized to G1/S border by double thymidine block (DTB), and then released in regular medium for 5 hours to enrich S/G2 cells. Phosphorylation at the S/T-Q motif was assessed using the protocol described in (A). ( D ) Phosphorylation of RECQL4 at the S/T-Q motif is attenuated when DNA-PK cs is inhibited. HEK293T cells expressing 3XFLAG-tagged RECQL4 were pretreated with 10 μM NU7441 or DMSO for 2 h, mock-treated or irradiated for 10 Gy, and allowed to recover for 20 min. Samples were processed and analyzed as stated in (A). ( E ) DNA-PK phosphorylates RECQL4 in vitro . Purified 3XFLAG-tagged RECQL4 was treated with λPP, and incubated with DNA-PK cs , Ku70/80 and DNA in the presence of ATP. Phosphorylation at the S/T-Q motif was assessed with Western blotting. ( F ) DNA-PK cs -mediated phosphorylation sites on RECQL4. RECQL4 was phosphorylated by DNA-PK in vitro and six phosphorylation sites (S27, S101, T116, S180, S326 and T336) in the N-terminal region of RECQL4 were identified by mass spectrometry analysis. Sld2, Sld2-like domain; NLS, nuclear location signal; Helicase, RecQ helicase domain; Zn-binding, Zn 2+ binding motif; CTD, C-terminal domain. ( G ) Ablating the DNA-PK cs -mediated phosphorylation sites on RECQL4 results in a decrease in IR-induced phosphorylation of RECQL4 at the S/T-Q motif. HEK293T cells transiently expressing 3XFLAG-tagged wild-type RECQL4 (FLAG-RQ4) or phosphorylation-null mutant (FLAG-RQ4-6A) were mock treated or irradiated with 10 Gy and allowed to recover for 20 min. Samples were processed and analyzed as stated in (A). ( H ) DNA-PK cs -dependent phosphorylation of RECQL4 is significantly decreased when the six phosphorylation sites are ablated. Purified FLAG-RQ4 and FLAG-RQ4-6A were treated with λPP, eluted from the FLAG-M2 beads, and incubated with DNA-PK cs , Ku70/80 heterodimer and DNA in presence of ATP. Phosphorylation at the S/T-Q motif was assessed with Western blotting as described above.

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Irradiation, Purification, SDS Page, Western Blot, Phospho-proteomics, Knock-Out, Blocking Assay, In Vitro, Incubation, Mass Spectrometry, Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: DNA-PK cs -mediated phosphorylation of RECQL4 promotes NHEJ. ( A ) Ablating the DNA-PK cs -mediated phosphorylation sites on RECQL4 significantly reduces the recruitment of RECQL4 to laser-generated DSBs. Relative fluorescent intensity of GFP-tagged wild-type RECQL4 (WT) and phosphorylation-null mutant RECQL4 (6A) following micro-irradiation are presented as mean ± SEM from 9 WT and 15 6A cells. ( B ) Blocking DNA-PK cs -dependent RECQL4 phosphorylation sites attenuates recruitment of NHEJ factors to the chromatin fraction following DNA damage. U2OS RECQL4 knockout cells stably expressing GFP-tagged WT or 6A were mock treated or irradiated with 10 Gy and allowed to recover for 10 min. Subsequently, cytoplasmic, soluble nuclear, and chromatin fractions were isolated for immunoblotting to examine the recruitment of proteins listed in the figure to the chromatin after irradiation. ( C ) RECQL4 phosphorylation modulates the IR-induced interactions between RECQL4 and NHEJ factors. U2OS RECQL4 knockout cells expressing GFP-tagged WT or 6A RECQL4 were irradiated with 10 Gy and allowed to recover for 10 min. GFP-tagged RECQL4 proteins were immunoprecipiated using a GFP antibody, the samples were resolved via SDS-PAGE, and interactions between RECQL4 and the core NHEJ factors DNA-PK cs , Ku70 and XRCC4 was determined via immunoblotting. ( D ) Ablating DNA-PK cs -mediated phosphorylation sites on RECQL4 decreases the interactions between the core NHEJ factors. U2OS RECQL4 knockout cells expressing GFP-tagged WT or 6A RECQL4 were irradiated with 10 Gy and allowed to recover for 10 min. DNA-PK cs was immunoprecipiated, the samples were resolved via SDS-PAGE, and interactions between DNA-PK cs and the core NHEJ factors Ku70, XRCC4 and XLF and RECQL4 was determined via immunoblotting. ( E ) RECQL4 phosphorylation promotes NHEJ-mediated DSB repair. Endogenous RECQL4 was depleted using RECQL4 siRNAs in U2OS cells stabilizing the NHEJ GFP reporter assay EJ5 and subsequently the cells were transiently transfected with empty vector or siRNA-resistant plasmids that express 3XFLAG-tagged WT or 6A RECQL4. NHEJ-mediated DSB repair was evaluated using the GFP-based reporter assay. Student's t-test (two-sided) was performed to assess statistical significance (** P < 0.01). ( F ) IR-induced 53BP1 foci resolution is attenuated in 6A cells compared to WT in G1 cells. U2OS parental (U2OS) and RECQL4 knockout (RQ4KO) cells as well as RECQL4 knockout cells stably expressing wild type RECQL4 (RQ4KO + RQ4WT) or phosphorylation mutant (RQ4KO + RQ4-6A) were irradiated with 2 Gy of γ-rays and 53BP1 foci formation and resolution was assessed 0.5, 1, 3 and 7 h post-IR. Remaining 53BP1 foci at each time point were calculated in over 50 Cyclin A-negative cells and the data are presented as Mean ± SEM. Student's t-test (two-sided) was performed to assess statistical significance (**** P < 0.0001). ( G ) Colony formation assays were performed to compare the radiation sensitivities of U2OS cells, U2OS cells in which endogenous RECQL4 was depleted using RECQL4 siRNAs, and U2OS RECQL4 knockdown cells complemented with 3XFLAG-tagged WT or 6A RECQL4. Cells were left cycling, irradiated at the indicated doses, and plated for analysis of survival and colony-forming ability. Student's t-test (two-sided) was performed to assess statistical significance (* P < 0.05).

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Generated, Mutagenesis, Irradiation, Blocking Assay, Knock-Out, Stable Transfection, Expressing, Isolation, Western Blot, SDS Page, Reporter Assay, Transfection, Plasmid Preparation, Knockdown

Journal: Nucleic Acids Research

Article Title: DNA-PK cs -dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks

doi: 10.1093/nar/gkac375

Figure Lengend Snippet: Model for the involvement of RECQL4 in NHEJ. Following induction of a DSB, the DNA-PK complex binds to the DSB ends. RECQL4 is then recruited to the DSB and phosphorylated by DNA-PK cs on six residues in its N-terminus, which increases DNA-PK-mediated DNA end bridging. The ligase factors are recruited and RECQL4 in conjunction with DNA-PK stabilizes the NHEJ long range synaptic complex. Subsequently, DNA-PK cs dissociates, the DSB is ligated following formation of the short range synaptic complex, and the rest of the NHEJ machinery detaches following completion of NHEJ. The model was generated with BioRender.com.

Article Snippet: Knockdown of RECQL4 via siRNA (Supplemental Table 1) was performed as previously described ( ) and the control siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Generated