Fig. 3.

Phosphorylation of Sp1 by ATM is necessary for Sp1 degradation and damage-associated senescence. a–g hTert-BJ1 cells were depleted of Sp1 using CRIPSR/Cas9 and transduced with lentivirus expressing Flag-tagged Sp1^WT, Sp1^S101A, or Sp1^S101E (Supplemental Fig. 1a). a Cells were treated with 200 μM H₂O₂ for 2 h, then placed in fresh media for 24 h. Lysates were collected at indicated time points past H₂O₂ release and used for Western blot analysis of protein levels. b–g Cells were treated with 200 μM H₂O₂ for 2 h, then placed in fresh media for 7 days. b–e 6 h prior to fixation, cells were treated with 10 μM EdU. Cells were then fixed and stained for EdU, γH2Ax, and DAPI. Scale bar represents 400 μm. f Cells described above were also stained for β-galactosidase. Scale bar represents 200 μm. g RNA was collected from cells described above. Quantitative RT-PCR was used to analyze samples with verified primers for SASP markers. GAPDH was used as a reference gene. Data were processed using the ΔΔCt method. Data represent means and SEM from 3 independent experiments assessed in triplicate. Significant differences between groups were determined by two-tailed Student’s t test. *, **, or *** indicate p values < 0.05, 0.01, or 0.001, respectively. No * indicates p value > 0.05