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(A) Representative growth curves of MRC5 cells in 20% and 3% oxygen as in for WI38 cells. (B) Additional data on the effect of ATMi, ATRi, and Chk1i on the BrdU incorporation in WI38 cells grown to senescence in 3% oxygen analyzed as in . (C) and (D) Effect of the indicated concentrations of ATMi, ATRi, Chk2i, and Chk1i on the BrdU incorporation in MRC5 cells grown to senescence in 20% or 3% oxygen analyzed as in and . (E) and (F) Effect of Chk2i, ATRi, or Chki in combination with ATMi on BrdU incorporation in the indicated cells grown to senescence at 3% oxygen. (G) MRC5 growth curves with and without long-term ATMi1 (KU-60019, 670 nM) treatment at 3% and 20% oxygen. (H) MRC5 growth curves with two different ATMi1 concentrations (KU-60019, 670 nM and 1.34 μM) at 20% oxygen. (I) and (J) Effect of combinations of ATMi with Chk2i, ATRi, or Chk1i on the BrdU incorporation of WI38 (I) or MRC5 (J) cells that had grown to senescence in the presence of ATMi. Experiments represent parallel growth, treatment, and quantification with the cells in (E) and (F). (K) Western blots for the indicated shelterin subunits in uninfected MRC5 cells or those infected with TRF2, <t>Rap1,</t> TPP1/POT1, or a vector control as indicated. (L) Growth curves of MRC5 cells in (K). (M) 4-day BrdU incorporation experiment on MRC5 cells grown to senescence and treated in 20% or 3% oxygen, as indicated. Vector-infected or TRF2-overexpressing cells were treated with inhibitors of ATM, ATR or Chk1. Ordinary One-way ANOVA for (B), (C), and (D). Two-way ANOVA for all other panels. P values: ****, < 0.0001; ***, < 0.001; **, < 0.01; * < 0.05. ns, not significant. ATMi, KU-60019; ATMi2, AZD1390; ATRi1. AZD6738; ATRi2, AZ20; ATRi3, M4344; Chk1i, CHIR-124.
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(A) Representative growth curves of MRC5 cells in 20% and 3% oxygen as in for WI38 cells. (B) Additional data on the effect of ATMi, ATRi, and Chk1i on the BrdU incorporation in WI38 cells grown to senescence in 3% oxygen analyzed as in . (C) and (D) Effect of the indicated concentrations of ATMi, ATRi, Chk2i, and Chk1i on the BrdU incorporation in MRC5 cells grown to senescence in 20% or 3% oxygen analyzed as in and . (E) and (F) Effect of Chk2i, ATRi, or Chki in combination with ATMi on BrdU incorporation in the indicated cells grown to senescence at 3% oxygen. (G) MRC5 growth curves with and without long-term ATMi1 (KU-60019, 670 nM) treatment at 3% and 20% oxygen. (H) MRC5 growth curves with two different ATMi1 concentrations (KU-60019, 670 nM and 1.34 μM) at 20% oxygen. (I) and (J) Effect of combinations of ATMi with Chk2i, ATRi, or Chk1i on the BrdU incorporation of WI38 (I) or MRC5 (J) cells that had grown to senescence in the presence of ATMi. Experiments represent parallel growth, treatment, and quantification with the cells in (E) and (F). (K) Western blots for the indicated shelterin subunits in uninfected MRC5 cells or those infected with TRF2, Rap1, TPP1/POT1, or a vector control as indicated. (L) Growth curves of MRC5 cells in (K). (M) 4-day BrdU incorporation experiment on MRC5 cells grown to senescence and treated in 20% or 3% oxygen, as indicated. Vector-infected or TRF2-overexpressing cells were treated with inhibitors of ATM, ATR or Chk1. Ordinary One-way ANOVA for (B), (C), and (D). Two-way ANOVA for all other panels. P values: ****, < 0.0001; ***, < 0.001; **, < 0.01; * < 0.05. ns, not significant. ATMi, KU-60019; ATMi2, AZD1390; ATRi1. AZD6738; ATRi2, AZ20; ATRi3, M4344; Chk1i, CHIR-124.

Journal: bioRxiv

Article Title: Replicative senescence is ATM driven, reversible, and accelerated by hyperactivation of ATM at normoxia

doi: 10.1101/2024.06.24.600514

Figure Lengend Snippet: (A) Representative growth curves of MRC5 cells in 20% and 3% oxygen as in for WI38 cells. (B) Additional data on the effect of ATMi, ATRi, and Chk1i on the BrdU incorporation in WI38 cells grown to senescence in 3% oxygen analyzed as in . (C) and (D) Effect of the indicated concentrations of ATMi, ATRi, Chk2i, and Chk1i on the BrdU incorporation in MRC5 cells grown to senescence in 20% or 3% oxygen analyzed as in and . (E) and (F) Effect of Chk2i, ATRi, or Chki in combination with ATMi on BrdU incorporation in the indicated cells grown to senescence at 3% oxygen. (G) MRC5 growth curves with and without long-term ATMi1 (KU-60019, 670 nM) treatment at 3% and 20% oxygen. (H) MRC5 growth curves with two different ATMi1 concentrations (KU-60019, 670 nM and 1.34 μM) at 20% oxygen. (I) and (J) Effect of combinations of ATMi with Chk2i, ATRi, or Chk1i on the BrdU incorporation of WI38 (I) or MRC5 (J) cells that had grown to senescence in the presence of ATMi. Experiments represent parallel growth, treatment, and quantification with the cells in (E) and (F). (K) Western blots for the indicated shelterin subunits in uninfected MRC5 cells or those infected with TRF2, Rap1, TPP1/POT1, or a vector control as indicated. (L) Growth curves of MRC5 cells in (K). (M) 4-day BrdU incorporation experiment on MRC5 cells grown to senescence and treated in 20% or 3% oxygen, as indicated. Vector-infected or TRF2-overexpressing cells were treated with inhibitors of ATM, ATR or Chk1. Ordinary One-way ANOVA for (B), (C), and (D). Two-way ANOVA for all other panels. P values: ****, < 0.0001; ***, < 0.001; **, < 0.01; * < 0.05. ns, not significant. ATMi, KU-60019; ATMi2, AZD1390; ATRi1. AZD6738; ATRi2, AZ20; ATRi3, M4344; Chk1i, CHIR-124.

Article Snippet: Antibodies: TRF2, #647 ( ); TRF1, #371 ( ); Rap1, #765 ( ); TIN2, #864 ( ); TPP1, #1150 ( ); POT1, #978 ( ); POT1, Proteintech 10581-1-AP, Vinculin, CST 13901; GAPDH, Invitrogen MA5-15738; Mre11, CST 4895; Chk2, CST 2662; HIF-1α, Abcam ab179483; ATM, CST 2873; γ-tubilin, Sigma GTU88.

Techniques: BrdU Incorporation Assay, Western Blot, Infection, Plasmid Preparation, Control