Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

doi: 10.1165/rcmb.2014-0263OC

Figure Lengend Snippet: BTB and CNC homology 1, basic leucine zipper transcription factor (Bach1) binds to an antioxidant response element within DHS-44(279) in vitro. (A) Scanning mutagenesis identified the critical nucleotides for transcription factor binding at 44-a and 44-c. (B, C, and E) EMSA experiments with 32P-labeled 44-a (B), 44-c (C), or 44-I (E) probes and 16HBE14o- nuclear extract. The DNA–protein complex generated with each probe was competed by 50× excess of the same unlabeled oligonucleotide but not by mutant versions (44-a/mut and 44-c/mut) (A, Table E3). The complex formed with the 44-a probe was also competed by an unlabeled Bach1 consensus oligonucleotide (39, 40) (Table E3) but not by a mutant version (B). (D) Mutagenesis of DHS-44(279) decreases its enhancer activity, confirming critical transcription factor binding sites. pGL3 constructs and transient reporter gene assays in 16HBE14o- cells as described in Figure 1. Mutant plasmids (44-a/mut and 44-c/mut are shown in A; 44-b/mut is shown in Fig. 2A) were compared with wild-type DHS-44(279). Data show luciferase activities relative to the CFTR basal promoter vector (= 1). Error bars represent SEM (n = 6). *P < 0.05 using unpaired t tests. (E) Binding of Bach1 to 44-I in vitro. The DNA–protein complexes formed with 32P-labeled 44-I probe were competed by 50× excess of unlabeled 44-I and Bach1 consensus probe but not by mutant versions (Table E3). A supershift is seen on incubation of the DNA–protein complex with an antibody specific for Bach1 (NF-κB or nuclear factor, erythroid 2-like 2, isotype controls).

Article Snippet: Bach1 ( 28 ) and Nrf2 (36971; Addgene, Cambridge, MA) cDNAs were transfected into 16HBE14o- cells as described above and harvested after 48 hours.

Techniques: In Vitro, Mutagenesis, Binding Assay, Labeling, Generated, Activity Assay, Construct, Luciferase, Plasmid Preparation, Incubation

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

doi: 10.1165/rcmb.2014-0263OC

Figure Lengend Snippet: Sulforaphane (SFN) regulates CFTR expression in airway epithelial cells. (A) Western blot showing the relocation of nuclear factor, erythroid 2-like 2 (Nrf2) into the nucleus after 2 hours of SFN treatment in 16HBE14o- cells. (B and C) Two hours of SFN treatment increases CFTR mRNA levels in 16HBE14o- cells (B) and human bronchial epithelial cells (C). CFTR mRNA levels assayed by Taqman quantitative RT-PCR normalized to 18S RNA levels. (D) Recruitment of Bach1, Nrf2, and with v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) to DHS-44(279) during 6 hours of SFN treatment in 16HBE14o- cells. ChIP with antibodies specific for each factor is shown after 0, 2, 4, and 6 hours of SFN treatment. Data were normalized to IgG and combined from at least two ChIP experiments. In all panels, error bars represent SEM. *P < 0.05 using an unpaired t test.

Article Snippet: Bach1 ( 28 ) and Nrf2 (36971; Addgene, Cambridge, MA) cDNAs were transfected into 16HBE14o- cells as described above and harvested after 48 hours.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

doi: 10.1165/rcmb.2014-0263OC

Figure Lengend Snippet: Overexpression of Bach1 and Nrf2 alters CFTR expression levels and the activity of the DHS-44kb element, where β-tubulin is the normalizer. (A) Western blots show overexpression of Nrf2 and Bach1 in whole cell lysate from 16HBE14o- cells. (B) After SFN treatment, overexpression of Nrf2 and Bach1 influences CFTR expression in 16HBE14o- cells. Nrf2 or Bach1 expression plasmids or vector alone (pcDNA3.1) were transfected into 16HBE14o- cells. After 48 hours, the cells were treated with 10 μM SFN for 0, 2, and 4 hours after cell harvest. CFTR mRNA levels assayed by Taqman quantitative RT-PCR (normalized to 18 s rRNA) are shown: pcDNA3.1 control (white bar), Nrf2 overexpression (gray bars), and Bach1 overexpression (black bars). Error bars represent SEM (n ≥ 6). *P < 0.05 using an unpaired t test. (C) Overexpression of Nrf2 influences the enhancer activities of DHS-44(279) in 16HBE14o- cells. pGL3 245 or pGL3 245 DHS-44(279) were cotransfected with Nrf2 or Bach1 expression plasmids or pcDNA3.1. After 48 hours, the cells were treated with 10 μM SFN for 0, 2, and 4 hours before cell harvest. Data show the relative enhancer activity of DHS-44(279) compared with the CFTR promoter (pGL3 245) when cotransfected with pcDNA3.1 (white bars) or Nrf2 (gray bars) followed by SFN treatment or Bach1 without SFN treatment (black bars). Error bars represent SEM (n ≥ 6). *P < 0.05 using an unpaired t test. ns, not significant.

Article Snippet: Bach1 ( 28 ) and Nrf2 (36971; Addgene, Cambridge, MA) cDNAs were transfected into 16HBE14o- cells as described above and harvested after 48 hours.

Techniques: Over Expression, Expressing, Activity Assay, Western Blot, Plasmid Preparation, Transfection, Quantitative RT-PCR, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Oxidative Stress Regulates CFTR Gene Expression in Human Airway Epithelial Cells through a Distal Antioxidant Response Element

doi: 10.1165/rcmb.2014-0263OC

Figure Lengend Snippet: Distal cis-regulatory elements interact to regulate CFTR expression in the airway. (A) When combined in tandem in the same vector DHS −35 kb and −44 kb have a modest cooperative effect on enhancer activity in vitro. 16HBE14o- cells were transfected with pGL3B luciferase-reporter constructs containing the CFTR basal promoter and fragments at DHS −44 kb or −35 kb individually or combined in the enhancer site in the forward orientation. Enhancer activities are shown relative to the CFTR basal promoter–alone vector (= 1); error bars represent SEM (n = 6). *P ≤ 0.05 when the single fragments are compared with the combined fragments using unpaired t tests. (B) siRNA-mediated knockdown of nuclear factor Y (NF-Y) enhances CFTR expression in 16HBE14o- cells (from Ref. 22). Western blot shows the efficiency of nuclear transcription factor Y, subunit A (NF-YA) depletion by siRNA in comparison to nontargeting control (NC) in 16HBE14o- cells. Cells were lysed after 72 hours to evaluate CFTR mRNA expression by Taqman quantitative RT-PCR from total RNA as described in Figure 4 legend. Error bars represent SEM (n ≥ 9). *P < 0.001 using an unpaired t test. (C) NF-YA depletion alters Nrf2 enrichment at DHS-44kb in 16HBE14o- cells. Cells were reverse transfected for 72 hours with siRNA targeting human NF-YA (KD [knockdown], gray bars) or negative control (NC, white bars). ChIP with antibodies specific for Nrf2 followed by quantitative PCR analysis. Primers (Table E3) are located at multiple DHS 5′ to the CFTR promoter. Data normalized to IgG are from a single representative ChIP experiment (the experiment was performed twice). Error bars represent SEM. *P < 0.05 using an unpaired t test.

Article Snippet: Bach1 ( 28 ) and Nrf2 (36971; Addgene, Cambridge, MA) cDNAs were transfected into 16HBE14o- cells as described above and harvested after 48 hours.

Techniques: Expressing, Plasmid Preparation, Activity Assay, In Vitro, Transfection, Luciferase, Construct, Knockdown, Western Blot, Comparison, Control, Quantitative RT-PCR, Negative Control, Real-time Polymerase Chain Reaction

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