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1 Supporting Information Identification of Novel ROS Inducers: Quinone Derivatives Tethered to Long Hydrocarbon Chains Yeonsun Hong,, Sandip Sengupta,, Wooyoung Hur, *, Taebo Sim,, * KU-KIST Graduate School of Converging Science and Technology, 145 Anam-ro, Seongbuk-gu, Seoul, , Republic of Korea Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), 5 Hwarangro 14-gil, Seongbuk-gu, Seoul, , Republic of Korea These authors contributed equally. Table of Contents: Supplementary protocol S2 Figure S1: Measurement of cellular GSH and GSSG levels in A549 cells following compound treatment for 4 hours. S3 Figure S2: FACS analysis showed apoptosis in A549 and HCT-116 cells after treatment of for 48 hours S4 Figure S3: Soft agar assay using A549 cells S5 S1

2 Supplementary protocol Reagents A549 lung cancer cells, HCT-116 colon cancer cells, A375 melanoma, SK-MEL-2 melanoma cells, and HFF-1 fibroblast cells were purchased from ATCC. NCI-H1299 lung cancer cells were purchased from Korean Cell Line Bank. and N-acetyl cysteine were from Sigma Aldrich. Antibodies for PARP, AIF, caspase-3, actin were purchased from Santa Cruz Biotech. The other antibodies used for Western blot analysis were from Cell Signaling Technology. Cell culture A549, HCT-116, and SK-MEL-2 cells were grown in RPMI-164 medium (Welgene, Korea) containing 1% FBS (Welgene, Korea) and 1% antibiotics. A375, HaCaT, MCF1A, and HFF-1 cells were grown in DMEM medium (Welgene, Korea) containing 1% FBS and 1% antibiotics. NCI-H1299 cells were grown in RPMI-164 medium supplemented with HEPES (Welgene, Korea), sodium pyruvate, 1% FBS and 1% antibotics. Annexin V-FITC-PI (FACS) analysis: Cells were seeded 6x1 5 cells/well in a 6 mm dish, and incubated overnight. Cells were then treated with the compounds for 48 hours at 37 o C in a 5% CO 2 atmosphere. Total cells were harvested using trypsin-edta solution, collected by centrifugation, washed with PBS and resuspended in buffer. Cells were stained with Annexin V (1:3) for 3 minutes, followed by PI (1:4) staining for 5 minutes. Fluorescence intensity was analyzed using a flow cytometer (BDCAnto II, BD Biosciences, USA). Measurement of cellular GSH, GSSG levels Cells were seeded at 1x1 4 cells/well in a white 96-well plate and incubated for hours. Cells were then treated with the compounds for 3 hours and subjected to GSH-GSSG-glo assay kit (Promega) following manufacturer s procedure. Luminescence intensity was detected using a microplate reader (Perkin Elmer). Soft agar assay In a 6-well plate, a mixed solution of 1 ml of 1% melting agarose and 1 ml of 2X RPMI containing 2% FBS and 2x antibiotics was poured. After cooling the agar solution, 1 ml of.6% melting agarose and 1 ml of A549 cells (1x1 4 cells/ml) in 2X RPMI containing 2% FBS and 2x antibiotics were mixed and plated on the bottom agar. The compounds were then added to the top agar. The medium was changed in 2 times per week. After incubation for 1 days, the agar was stained with.5% crystal violet for 2 hours. Each experiment was tested in duplicate. The colonies were observed by microscope and photographed using in X mode and analyzed by Image J software. S2

3 Total GSH GSSG Reduced GSH Supplementary Figure * Figure S1. Measurement of cellular GSH and GSSG levels in A549 cells following 1 M compound treatment for 4 hours. Compound slightly diminished reduced GSH level mainly due to GSH oxidation, whereas and piperlongumine significantly depleted the total level of GSH, indicating and piperlongumine have another mode for apoptosis other than ROS generation. Compared with piperlongumine, further depleted the total GSH level, which might be associated with its stronger cytotoxicity. Each data represents a mean value with standard deviation from sextuplicated points and was analyzed by one-way ANOVA analysis. *p <.1, p <.1 S3

4 Figure S2. FACS analysis showed apoptosis in A549 and HCT-116 cells after treatment of for 48 hours. S4

5 Figure S3. Soft agar assay using A549 cells. Each compound was treated at 1 M for 1 days. Piperlongumin and significantly decreased the colony size, while more profoundly reduced it than both piperlongumine and, indicating that and were effective in blocking anchorage-independent A549 cell growth in vitro. S5

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