Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: The effects of siRNA knockdowns of Polθ and other TLS Pols on replicative bypass of a cis-syn TT dimer or a (6–4) TT photoproduct carried on the leading or lagging DNA strand template in XPA human fibroblasts

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques:

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: UV induced mutation frequencies in the cII gene in BBMEF cells expressing a (6–4) PP photolyase,CPD photolyase, or no photolyase and treated with siRNA for Polθ or other TLS Pols

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques: Mutagenesis, Expressing, Plasmid Preparation

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: (A) Schematic of DNA fiber assay and representative images of stretched DNA fibers in UV damaged GM637 HFs treated with control (NC), Polη, Polθ, or Polη and Polθ siRNAs

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques: Control

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: (A) Schematic for targeting the knock outs of Polη and Polθ genes and RT-PCR analyses of Polη−/−, Polθ−/− and Polη−/− Polθ−/− MEFs. GAPDH was used for a negative control.

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques: Reverse Transcription Polymerase Chain Reaction, Negative Control

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: (A) BrdU immuno-assay for ssDNA detection in UV irradiated or unirradiated MEFs. Cells were treated with BrdU for 20h and irradiated with UV (20 J/m2) or not, followed by 6h incubation. Immuno-staining with BrdU was visualized by fluorescence microscopy. (Left) representative images of BrdU staining in unirradiated or UV irradiated primary WT, Polθ−/−, Polη−/−, and Polη−/− Polθ−/− MEFs; (Right) quantification of BrdU immuno-staining intensity in unirradiated and UV irradiated primary MEFs. The mean and standard deviation were analyzed from 4 independent experiments and are indicated by a horizontal and a vertical black bar, respectively. Student’s two-tailed t-test values, ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques: Immuno Assay, Irradiation, Incubation, Immunostaining, Fluorescence, Microscopy, BrdU Staining, Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: (A) SCEs in unirradiated MEFs. (Left), representative images of metaphases in unirradiated primary WT, Polθ−/−, Polη−/−, and Polη−/− Polθ−/− MEFs. (Right), quantification of SCE frequency in unirradiated primary WT, Polθ−/−, Polη−/− and Polη−/− Polθ−/− MEFs. Each datum point represents a single metaphase and ~1,000 metaphase chromosomes were analyzed. The mean and standard deviation were analyzed from 4 independent experiments and are indicated by a numeral and a vertical black bar, respectively. Student’s two-tailed t-test p values, *, p<0.05; ***, p<0.001; ****, p<0.0001.

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques: Standard Deviation, Two Tailed Test

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: (A) UVB-induced skin tumors on the dorsal area of Polθ−/− mice at 42 weeks of UV exposure.

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques:

Journal: Cell

Article Title: Error-prone replication through UV lesions by DNA polymerase θ protects against skin cancers

doi: 10.1016/j.cell.2019.01.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Full length (7776 bp) human Polθ cDNA was obtained from Addgene plasmid Repository (plasmid #:73132).

Techniques: Knock-Out, Staining, Transfection, Construct, Plasmid Preparation, Purification, DNA Ligation, DNA Purification, Proliferation Assay, Single Cell Gel Electrophoresis, Cell Culture, Software

Journal: Oncotarget

Article Title: High Myc expression and transcription activity underlies intra-tumoral heterogeneity in triple-negative breast cancer

doi: 10.18632/oncotarget.15891

Figure Lengend Snippet: ( A ) The impact of Myc siRNA knockdown on the SRR2 reporter activity was measured by luciferase activity in RU and RR cells derived from MDA-MB-231 and SUM-149 cells. The western blots below showed the Myc knockdown efficiency. ( B ) RU cells derived from both TNBC cell lines were transiently transfected with pcDNA3.3-Myc. pcDNA empty vector (EV) was included as a negative control. The impact of enforced transient transfection of MYC on the SRR2 reporter activity was measured by luciferase activity. The western blots below showed the MYC transfection efficiency. β-actin protein expression was used as a loading control of the experiment. ( C ) RU cells derived from both TNBC cell lines were stably transfected with MYC and EV (negative control). The SRR2 probe pull-down assay was performed to assess the Myc and SRR2 binding in RU- MYC and RU-EV cells. The western blots in the right panel showed the input of the pull-down assay. HDAC-1 was used as a loading control of the experiment. ( D ) ChIP-qPCR was performed in RU- MYC and RU-EV cells derived from MDA-MB-231 cells to determine the Myc binding to SRR2 DNA sequence. RR-EV cells were included as a control of the experiment. ( E ) The impact of Myc siRNA knockdown on the Myc binding to SRR2 DNA sequence was performed using ChIP-qPCR in RR cells derived from MDA-MB-231 cells. RU cells transfected with scrambled siRNA were included as a control of the experiment. ( F ) The relative mRNA level of CCND1 was measured by qRT-PCR after Myc knockdown in RU and RR cells derived from MDA-MB-231 cells. The mRNA expression level was normalized with GAPDH. ( G ) The relative mRNA level of Myc transcript targets, CCND1 and TERT was measured by qRT-PCR in RU- MYC and RU-EV cells derived from MDA-MB-231 cells. The mRNA expression levels were normalized with GAPDH.

Article Snippet: The set of JASPAR vertebrate core set of transcription factors (Downloaded April 17, 2013) was applied to the reporter sequence on both strands with a p-value < 0.001. pcDNA3.3-Myc plasmid was a gift from Derrick Rossi (Addgene plasmid #26818) and pcDNA plasmid was purchased from Addgene (Cambridge, MA).

Techniques: Knockdown, Activity Assay, Luciferase, Derivative Assay, Western Blot, Transfection, Plasmid Preparation, Negative Control, Expressing, Control, Stable Transfection, Pull Down Assay, Binding Assay, ChIP-qPCR, Sequencing, Quantitative RT-PCR