Supp. Figs. 1-12 from Constitutive Activation of DNA Damage Checkpoint Signaling Contributes to Mutant p53 Accumulation via Modulation of p53 Ubiquitination

<p>Supplementary figures (1-12) Supplemental Fig. 1. Normalized levels of monoubiquitinated mutant p53 are shown for indicated cell lines analyzed in Fig. 1a. Supplemental Fig. 2. Longer single exposure of p53 blot in Fig. 2a is displayed to demonstrate the relative abundance of monoubiquitinated to native species. Supplemental Fig 3. (a) WI38 cells were transfected with p53-273H and MDM2 for 40- 48 hours later, and plates were treated with 2 μM Dox for 2 hours in the presence or absence of 2 mM caffeine. Caffeine-treated samples were pre-treated with 2 mM caffeine for 20 min prior to addition of Dox. Supplemental Fig. 4. Normalized levels of monoubiquitinated or native mutant p53 from p53-273H-expressing H1299 cells analyzed in Fig. 2c are shown. Supplemental Fig. 5. Normalized levels of monoubiquitinated and native mutant p53, as well as MDM2, are shown for ABC1-shGFP and ABC1-shMDM2 cells analyzed in Fig. 3a. Supplemental Fig. 6. Normalized levels of monoubiquitinated or native mutant p53 are shown from p53-273H-expressing H1299 cells co expressing wt vs. RING mutated MDM2 analyzed in Fig. 3b. Supplemental Fig. 7. Normalized levels of monoubiquitinated or native mutant p53, as well as p53 S15-p, are shown for p53-273H-expressing H1299 cells analyzed in Fig. 4a. Supplemental Fig. 8. Normalized levels of polyubiquitinated p53 mutant p53 are shown for H1048 cells analyzed in Fig. 5b. Supplemental Fig. 9. Normalized levels of polyubiquitinated p53 are shown in p53- 273H-expressing H1299 cells analyzed in Fig. 5c after mock, Dox or Dox+caffeine treatment. Supplemental Fig. 10. (a) H1048 cells were mock-treated or treated with caffeine and/or MG132 for 7 hours and fixed and analyzed by flow cytometry for p53. (b) ABC1 shGFP or shMDM2-expressing cells were treated with or without caffeine, and stained for p53 level by flow cytometry. Supplemental Fig 11. (a) H1975, (b) MDA MB 231, (c) WI38, or d) U2OS cells were mock-treated or treated with caffeine for 7 hours and fixed and analyzed by flow cytometry for p53. Supplemental Fig. 12. The level of total ATM in siATM or control siRNA transfected H1048 cells is shown. Actin was immunoblotted as a loading control.</p>