hct116 p53 knockout ko  (ATCC)

Journal: Redox Biology

Article Title: OGG1 co-inhibition antagonizes the tumor-inhibitory effects of targeting MTH1

doi: 10.1016/j.redox.2020.101848

Figure Lengend Snippet: Co-depletion of OGG1 in A549 lung adenocarcinoma cells protects against MTH1 depletion-induced DNA breaks and p53-induced senescence. A. Simultaneous lentiviral co-transduction and selection schemes employed in this experiment. The pLKO.shMTH1 construct is under hygromycin (h) selection and the pLKO.shOGG1 construct is under puromycin (p) selection. Control samples consist of co-transduced puromycin- and hygromycin-selectable pLKO.shGFP constructs. B. Immunoblot showing protein levels of OGG1 and MTH1 under the various individual or co-selection schemes denoted in (A) in the shRNA co-transduced cells. Actin is shown as the loading control. C. Quantitation of actin-normalized OGG1 and MTH1 protein expression levels from (B) under the different selection schemes. Normalized quantifications are based on pixel densities of immunoblot bands obtained via ImageJ. D. Alkaline comet assay. Representative olive tail moments (10× magnification) are indicated for the types of comets that were scored (no/medium/long tails) in the indicated cultures. Error bars (±SD) and p-values from an unpaired Student t -test are shown from two independent experiments. E. Senescence-associated beta-galactosidase (SA-beta-gal) activity assay. Cell senescence was assessed in the indicated cultures. Positive staining was quantified in approximately 50 cells per condition. Data represent two experimental replicates. Error bars (±SD) and p-values from an unpaired Student's t -test significance are shown. F. Immunoblot showing p53 and actin (loading control) protein levels in the indicated samples.

Article Snippet: The HCT116 p53 wildtype (wt) and p53 knockout (KO) isogenic human colon cancer cells were obtained from Dr. Bert Vogelstein's laboratory (Johns Hopkins University, Baltimore, MD).

Techniques: Transduction, Selection, Construct, Control, Western Blot, shRNA, Quantitation Assay, Expressing, Alkaline Single Cell Gel Electrophoresis, β-Gal Activity Assay, Staining

Journal: Redox Biology

Article Title: OGG1 co-inhibition antagonizes the tumor-inhibitory effects of targeting MTH1

doi: 10.1016/j.redox.2020.101848

Figure Lengend Snippet: Mitigation of DNA strand breaks and proliferative arrest observed upon MTH1 depletion in p53-null cells correlates with low OGG1 expression. A. Loss of p53 in A549 cells corresponds to decreased OGG1 levels. Immunoblotted total protein lysates were probed with the indicated antibodies. Tubulin is shown as the loading control. B. Loss of p53 in HCT116 colorectal cells corresponds to decreased OGG1 levels. Isogenic HCT116 cells either wildtype (wt) or null (KO) for p53 were immunoblotted against the indicated antibodies with GAPDH as the loading control. C. HCT116 cells lacking p53 do not express the cell cycle arrest marker, p21 cip1 upon MTH1 depletion. Immunoblotted total protein lysates were probed with the indicated antibodies. GADPH is shown as the loading control. D. Alkaline comet assay. Percentage of cells with no tails (as in E) vs. those with DNA tails (indicating DNA breaks) are graphed for the indicated samples. Note we did not observe the variations in DNA olive tail moments seen for A549 ( E) to warrant separation of HCT116 cells into ‘medium’ and ‘long’ tails. Error bars (± SD) and p-values from an unpaired Student t test are shown from two independent experiments.

Article Snippet: The HCT116 p53 wildtype (wt) and p53 knockout (KO) isogenic human colon cancer cells were obtained from Dr. Bert Vogelstein's laboratory (Johns Hopkins University, Baltimore, MD).

Techniques: Expressing, Control, Marker, Alkaline Single Cell Gel Electrophoresis

Journal: Redox Biology

Article Title: OGG1 co-inhibition antagonizes the tumor-inhibitory effects of targeting MTH1

doi: 10.1016/j.redox.2020.101848

Figure Lengend Snippet: Small molecule-based co-inhibition of OGG1 and MTH1 does not exhibit consistent enhanced cytotoxicity in tumor cells vs. individual OGG1 or MTH1 inhibitor treatment. A. Published chemical structures of SU0268 (OGG1 inhibitor), SU0383 (MTH1/OGG1 dual inhibitor), IACS-4759 (MTH1 inhibitor). Each shaded square represents the corresponding treatment condition data graphed for the viability assay in (B). B. Normalized cell viability. A549 cells were dosed, in triplicate, with SU0268, SU0383, IACS-4759 or SU0268/IACS-4759 at the indicated concentrations. Vehicle represents DMSO. Dose response was evaluated at 24, 48, and 72 h following treatment via the vehicle-normalized luminescence signal in a Cell Titer-Glo® assay. Error bars (±SD) and p-values are indicated within the graphs (*p < 0.05, **p < 0.01). Results shown are representative of two independent experiments. C. Normalized cell viability. H460 cells were dosed, in triplicate, with SU0268, SU0383, IACS-4759 or SU0268/IACS-4759 at the indicated concentrations. Vehicle represents DMSO. Dose response was evaluated at 48 and 72 h following treatment via the vehicle-normalized luminescence signal in a Cell Titer-Glo® assay. Error bars (±SD) and p-values are indicated within the graphs (*p < 0.05, **p < 0.01). D. Normalized cell viability. HCT116 cells were dosed, in triplicate, with SU0268, SU0383, IACS-4759 or SU0268/IACS-4759 at the indicated concentrations. Vehicle represents DMSO. Dose response was evaluated at 48 h following treatment via the vehicle-normalized luminescence signal in a Cell Titer-Glo® assay. Error bars (±SD) and p-values are indicated within the graphs (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: The HCT116 p53 wildtype (wt) and p53 knockout (KO) isogenic human colon cancer cells were obtained from Dr. Bert Vogelstein's laboratory (Johns Hopkins University, Baltimore, MD).

Techniques: Inhibition, Viability Assay, Glo Assay