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1 Supporting Information Bauer et al /pnas SI Materials and Methods Immunofluorescence experiments were performed as described (8). Briefly, NUGC-3 cells were treated with 25 μm PK11007 or DMSO control for 2 or 4 h, fixed with 4% (vol/vol) paraformaldehyde, and permeabilized with 0.5% (wt/vol) Triton X-100. The following antibodies were used: anti-p53 antibody Pab 1620 (Abcam) and anti-p53 antibody Pab 240 (Abcam). Hoechst (Cell Signaling) stain was used to stain the nucleus of cells. Imaging was performed using a Leica TCS SP8 confocal microscope. Fig. S1. DSF ΔT m values of different p53 mutants (8 μm) after incubation with diverse 2-sulfonylpyrimidine compounds (250 μm) for 30 min. 1of9

2 Fig. S2. 15 N- 1 H HSQC NMR spectrum of the p53 Y220C core domain (red) with 1,000 μm (blue), 436 μm (yellow), and 218 μm (green) PK11000 at 293 K. Fig. S3. Alkylation of full-length p53 with 2-sulfonylpyrimdines does not compromise its DNA binding capability. Stabilized full-length p53 was incubated with 1 mm PK11000, PK11007, PK11010, and 5% DMSO for 2 h at 4 C and then titrated into a cuvette containing 20 nm carboxyfluoresceine-labeled Gadd45a DNA. Fluorescence polarization data were recorded and fitted to the Hill equation including a linear drift term. 2of9

3 Fig. S4. Cell viability time course for (A) HUH-6 (p53 WT), HUH-7 (p53 Y220C), and (B) MKN1 cancer cells at 30 or 60 μm PK11007(10and30μM for MKN1). Cell viability was measured in quadruplicates and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM. 3of9

4 Fig. S5. Biological effects of 2-sulfonylpyrimidines on diverse cell lines. Concentration-dependent viability reduction of WI-38 (p53 WT, noncancer), NUGC-4 (p53 WT), HUH-6 (p53 WT), SJSA-1 (p53 WT), SW480 (p53 R273H/P309S), NUGC-3 (p53 Y220C), HUH-7 (p53 Y220C), and MKN1 (p53 V143A) cells after compound treatment for 24 h. The strongest effects on cell viability were observed for PK11007 and PK Mutant p53 cell lines were mostly significantly more sensitive for these compounds than p53 WT and noncancer cell lines. This behavior was also observed for PK11010 and PK11029, however only at higher compound concentrations. PK11003 led to a strong viability decrease; however, it did not distinguish clearly between mutant and WT p53 cell lines. Compounds that contain a negatively charged carboxyl group, such as PK11000 and PK11009, did not lead to a significant cell viability decrease in the tested cell lines at concentrations below 120 μm. Mutant p53 cell lines were also extra sensitive to compounds that destabilized the p53 core domain in vitro, such as PK11012 and PK Cell viability was measured in quadruplicate and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM. 4of9

5 Fig. S6. Viability reduction of APR-246 in NUGC-3 (p53 WT), HUH-6 (p53 WT), NUGC-4 (p53 Y220C), and HUH-7 (p53 Y220C) cancer cells after treatment for 24 h. Cell viability was measured in quadruplicates and normalized with the values of blank (viability = 1) and no cell (viability = 0) controls. Data are shown as mean ± SEM. 5of9

6 Fig. S7. PK11007 induces cell death via caspase-independent pathways. HUH-6 and HUH-7 cells were treated for 6 h with PK11007, APR-246, and Nutlin-3. (A) PK11007 and APR-246 treatment did not significantly increase caspase 3 and caspase 7 activities, only Nutlin-3 yielded a significant caspase induction in HUH-6 cells. (B) Membrane permeabilitywassignificantlyincreased for high PK11007 concentrations or incombination with BSO in HUH-7cells, whereas HUH-6 cells were not affected. APR-246 and Nutlin-3 did not significantly induce cytotoxicity. (C) PK11007 slightly increases caspase 3/7 activity in SW480 (at 15 μm), SJSA-1 (at 30 μm), and HCT116 (at 60 μm) cancer cells. All samples were measured in quadruplicate, with error bars depicting the SEM. Significance levels were calculated using a one-way ANOVA with the Bonferroni post hoc test for mean comparison (***P < 0.001; **P < 0.01; *P < 0.05). 6of9

7 Fig. S8. NAC prevents PK11007-mediated ROS formation. (A) Determination of intracellular ROS levels via CellROX Deep Red fluorescence after incubating HUH-6 and HUH-7 cancer cells with 5 mm NAC, 400 μm tert-butyl hydroperoxide (TBHP), or a combination of 30 μm PK11007 and 5 mm NAC for 1.25 h. NAC does not only decrease basal ROS levels, but also completely prevents PK11007 from increasing ROS levels. (B) Relative intracellular ROS levels via CellROX Deep Red fluorescence after incubating four cancer cell lines with 30 or 60 μm PK11007 for 2 h. PK11007 led to an increase of ROS in all tested cell lines; however, at high doses the relative ROS level was significantly higher in HUH-7 and NUGC-3 cells, whereas the ROS levels in MKN1 cells remained on the level of the p53wt cell lines. Median fluorescence levels were determined in triplicates. Error bars depict SEM values. Fig. S9. PK11007 changes the folding state of mutant p53 (Y220C) in NUGC-3 cells. PK11007 treatment led to a significant reduction of unfolded p53 (Pab 240) and a slight increase in folded p53 (Pab 1620). Hoechst dye was used to stain the nucleus. To facilitate comparison of Pab 1620 and Pab 240 fluorescence levels, we increased exposure of all raw pictures by 2 ev. Fig. S10. Protein levels of p53 after treatment with nontarget or p53 sirna in MKN1, HUH-6, and HUH-7 cancer cells. 7of9

8 Table S1. Description of cell lines Cell line p53 status Organism Tissue type Disease ATCC/JCRB code WI-38 WT Human Lung CCL-75 HUH-6 WT Human Liver Hepatoblastoma JCRB0401 NUGC-4 WT Human Stomach Gastric adenocarcinoma JCRB0834 SJSA-1 WT Human Fibroblast (lung) Osteosarcoma CRL-2098 HCT-116 WT Human Colon Colorectal carcinoma CCL-247 SW480 R273H-P309S Human Colon Dukes type B, colorectal adenocarcinoma CCL-228 HUH-7 Y220C Human Liver Hepatocellular carcinoma JCRB0403 NUGC-3 Y220C Human Stomach Gastric adenocarcinoma JCRB0822 MKN1 V143A Human Stomach Adenosquamous carcinoma JCRB0252 H1299 Null Human Lung Non small cell lung cancer CRL-5803 ATCC, American Type Culture Collection; JCRB, Japanese Collection of Research Bioresources. Table S2. Somatic mutations in antioxidative genes of tested cell lines Protein family Abbreviation NUGC-3 NUGC-4 HUH-7 HUH-6 SJSA-1 MKN1 SW480 H1299 HCT116 Glutathione peroxidases GPX GPX4 (c.325-2a > G)* Glutathione S-transferases GST GSTK1 (p.m100i) GSTM2 (p.n59s) GSTA2 (c.273-3c > T; c.249g > T);GSTP1 (G208V)* Thioredoxin-containing proteins TXN TXNDC11 (p.n440n; p.n467n) TXN2 (p.k152n); TXNDC5 (p.f368s); TXNDC12 (p.l7l); TXNRD1 (c.1_2inst; c.451_452inst)* Peroxiredoxins PRDX Superoxide dismutases SOD Catalase CAT Glutaredoxins GLRX Glutamate-cysteine GCL ligase Glutaminase GLS2 Glutathione synthetase GSS Gene mutation data 2, , ,100 *Mutation syntax follows the Human Genome Variation Society nomenclature recommendations (2). Data taken from the COSMIC Cell Lines Project. Data taken from the COSMIC database. 8of9

9 Table S3. Variable X-ray data collection and refinement statistics Value Data collection Space group P a, b, c, Å 65.11, 71.08, Molecules per asymmetric unit 2 Resolution (Å)* ( ) Unique reflections 92,560 Completeness (%)* 99.8 (99.5) Multiplicity* 5.5 (5.3) R merge (%)*, 6.4 (57.2) <I/σ I >* 17.4 (3.9) Wilson B value, Å Refinement No. of protein atoms 3,136 No. of water atoms 541 No. of zinc atoms 2 No. of ligand atoms 20 Overall B value, Å R cryst,% 17.8 R free,% 19.3 RMSD bonds, Å RMSD angles, 1.3 PDB ID code 5LAP *Values in parentheses are for the highest-resolution shell. R merge = P (I h,i <I h >)/ P I h,i. Number includes alternative conformations. R cryst and R free = P jjf obs j jf calc jj/ P jf obs j, where R free was calculated over 5% of the amplitudes chosen at random and not used in the refinement. 9of9

172R 172K TAM-2/172R TAM-2/172K. AZT concentration [nm] AZT concentration [nm] MgCl 2 2.5K 2.5K 5K 2.5K 5K 2.5K K 5K 2.5K 5K 2.5K 50 2.

172R 172K TAM-2/172R TAM-2/172K. AZT concentration [nm] AZT concentration [nm] MgCl 2 2.5K 2.5K 5K 2.5K 5K 2.5K K 5K 2.5K 5K 2.5K 50 2. 5 5 5 5 A MgCl 2 172R 172K TAM-2/172R TAM-2/172K AZT concentration [nm] B 172R 172K TAM-2/172R TAM-2/172K AZT concentration [nm] ATP + ATP - Supplemental Figure 1. Primer extension of HIV-1 RT polymorphisms

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