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Supporting Information Supporting Information Gupta et al. 10.1073/pnas.1525387114 SI Methods objective, and a Canon EOS 40D digital single-lens reflex cam- Chemical Approach for Dissection of the Breast TIC Program. Only era. Correlation analysis for G3BP2 and SART3 staining in the selected compounds that target MDA-MB-231 LM2 cells in adjacent sections then was performed to assess colocalization. combination with paclitaxel (0.2 μM for 48 h) were used. Of Image pairs were subjected to automated contrast enhancement 60,000 compounds screened, 256 were selected for the second and then were registered using Photoshop (Adobe). Any non- screening step. To eliminate toxic compounds, nonmalignant overlapping image areas created in the registration process were cells were treated with these small molecules. One hundred eliminated by cropping both images. Next, the images were seventeen nontoxic compounds were selected for further analy- subjected to correlation analysis using ImageJ (https://imagej. sis. Cell viability (MTT) assays of modified MDA-MB-231 can- nih.gov/). Using the LAB color space, the images were seg- cer cells with five different concentrations (0.12, 0.37, 1.11, 3.33, mented to create binary images of the stained regions (thresh- and 10 mol/L) were carried out. To determine which genes bind olds for pass were L: 0–154; A: 0–255; BN: 123–255). An to this compound, TurboBeads carboxy nanoparticles (Turbo- automated procedure then quantified the number of positive Beads) were conjugated to compound C108 for 20 min per the pixels in 100 × 100 pixel (23 × 23 μm) regions of interest placed ’ manufacturer s protocol, followed by overnight immunoprecipi- in a grid pattern over the images. The corresponding datasets for tation at 4 °C. Proteins from metastatic cancer cells were pulled the two images then were subjected to statistical analysis to de- down with nanoparticles conjugated to compound C108 and with termine the correlation coefficient and P value using Prism nanoparticles alone as a control. Purified proteins were sepa- (GraphPad). rated on an 8% agarose gel, and a few bands that bound to compound C108 but not to the control were cut out from the gel RNA-Binding Protein Immunoprecipitation. A RNA-binding protein and analyzed by mass spectrometry. Mass spectrometry analy- immunoprecipitation kit (Millipore) was used per the manufac- sis revealed 44 proteins that bound to compound C108. We turer’s protocol. BT-474 cells were treated with 0.3 μM paclitaxel obtained shRNA-expressing lentivirus for each pull-down pro- for 1 h. RIP buffer was used to collect protein, followed by im- tein and infected modified MDA-MB-231 cells. The MTT assays munoprecipitation overnight at 4 °C with G3BP2 antibodies- were performed with shRNA stable cell lines treated with non- lethal doses of paclitaxel. Only shRNA G3BP2 made cells sen- A/G magnetic beads. A magnetic separator was used to separate sitive to treatment with paclitaxel. To confirm that compound the beads from the buffer, and the beads were washed three – C108 binds to G3BP2, immunoprecipitation with magnetic times with RIP wash buffer. The RNA protein complex was nanoparticles bound to compound C108 and nanoparticles alone digested with proteinase K for 30 min at 55 °C, and RNA was (control) was carried out. purified with a phenol:chloroform:isoamyl alcohol extraction. mRNA was purified with the RNAeasy Kit (Qiagen) and was Histologic Analysis of Clinical Samples. Adjacent tissue sections analyzed with the Affymetrix microarray. were stained for G3BP2 and SART3, respectively, and RGB Only mRNA data that showed an in increase of 1.6 or higher images were acquired using an Olympus BX40 microscope, 10× after treatment compared with the controls were selected. Gupta et al. www.pnas.org/cgi/content/short/1525387114 1of11 Fig. S1. Identification of anti-TIC chemical compounds. (A) Schematic illustration of screening approaches to identify anti-TIC chemical compounds. Of the 60,000 compounds screened, we selected 117 for the second screening step. We studied their efficacy at five different concentrations (0.12, 0.3, 1.11, 3.33, and 10 M). We then selected 78 of these 117 compounds that showed dose-dependent repression of cancer cell growth and that were nontoxic to normal human umbilical vein endothelial cells. (B) The molecular structures of the selected compounds. Gupta et al. www.pnas.org/cgi/content/short/1525387114 2of11 Fig. S2. G3BP2 rescues the decrease of ALDEFLUOR phenotype mediated by compound C108. (A) Magnetic beads were combined with compound C108 followed by immunoprecipitation of proteins to compound C108. Interactive proteins were analyzed by mass spectroscopy. The construction of stable cell lines with repressed interacting proteins was followed by the detection of paclitaxel-sensitive cells. (B) MDA-MB-453 cells show a G3BP2-mediated rescue of the decrease in ALDEFLUOR phenotype induced by C108+paclitaxel. Cells were treated with vehicle or with 1 μM compound C108 plus 0.1 μM paclitaxel or with 1 μM compound C108 plus 0.1 μM paclitaxel plus endogenous G3BP2 and were analyzed by flow cytometry. Gupta et al. www.pnas.org/cgi/content/short/1525387114 3of11 Fig. S3. Correlations of G3BP2 expression and outcomes of breast cancer patients. (A–C) The Kaplan–Meier Plotter was used to analyze the correlation of G3BP2 with outcomes of breast cancer patients. Division by the median showed worse recurrence-free survival (A), distant metastasis-free survival (B), and overall survival (C). (D–H) Increased G3BP2 levels correlate with worse recurrence-free survival in different subtypes of breast cancer: basal type (D), + ER-negative (E), ER-positive (F), luminal A (G), and luminal B (H). (I) In patients with HER2 breast cancer, increased G3BP2 levels show improved survival. Gupta et al. www.pnas.org/cgi/content/short/1525387114 4of11 Fig. S4. Colocalization of G3BP2 and PABPC1. (A) Stable knockdowns of G3BP2 were created with shRNA in the MDA-MB-453, BT-474, and MDA-MB-231 cell lines. (B) MDA-MB-231 cells were treated with 1 μM of paclitaxel for 30–60 min and were stained with G3BP2 (red) and PABPC1 (green) antibodies. (C) BT-474 cells and BT-474 cells with G3BP2 down-regulation were treated with 1 μM of paclitaxel for 30–60 min, stained with G3BP2 (red) and PABPC1 (green) anti- bodies, and imaged by multispectral confocal imaging. Gupta et al. www.pnas.org/cgi/content/short/1525387114 5of11 A scr-shRNA shRNA G3BP2 scr-shRNA 50 cells 500 cells shRNA G3BP2 B scr-shRNA shRNA G3BP2 cells 4 10 cells 5 10 Fig. S5. G3BP2 knockdown has a pronounced effect on tumor cell self-renewal. (A) Significantly higher numbers of cells BT-474 cells with silenced G3BP2 expression were required to form tumors. (B) ELDA for the tumor-forming frequency of G3BP2 shRNA MDA-MB-453 cells and control scr-shRNA MDA-MB-453 cells in NOD-SCID mice. Fig. S6. Down-regulation of G3BP2 SG protein in human breast cancer cells leads to a decrease of SART3. (A) Fluorescent immunocytochemical staining was performed to determine the expression of SART3 in G3BP2-depleted and control cells. (Magnification: 200×) (Scale bars, 20 μm.) (B) Immunocytochemistry was performed to determine the expression of SART3 in G3BP2-depleted and control cells. (Magnification, 200×.) (Scale bar, 20 μm.) Gupta et al. www.pnas.org/cgi/content/short/1525387114 6of11 A MDA-MB-453 B scr-shRNA shRNA SART3 ) (µm spheres Number of S Sphere size BT-474 ) (µm spheres Number of Sphere size Fig. S7. G3BP2 regulates Oct-4, Nanog, and SART3 expression. (A) Western blot analysis was performed to detect OCT-4 and Nanog expression in SART3- knockdown breast cancer cell lines. (B) Representative images and analysis of mammosphere formation observed in SART3-silenced (white bars) and control (black bars) BT-474 and MDA-MB-453 cells. Data represent mean ± SD; *P < 0.05; **P < 0.005. (Magnification: 200×.) Fig. S8. No change in vimentin or E-cadherin levels is seen after repression of G3BP2 in BT-474 cells. The effect of G3BP2 silencing on EMT markers was assessed using Western blot analysis. No change in vimentin or E-cadherin was noted. TWIST1 and Slug protein levels were decreased after knockdown of G3BP2. Gupta et al. www.pnas.org/cgi/content/short/1525387114 7of11 Table S1. Proteins that bind to anticancer stem-cell compound C108 Protein coverage determined N Protein Protein name Matches by amino acid count, % 1 SNW1 SNW domain-containing protein 1 17 201/536 37.5 2 USP39 U4/U6-snRNP-associated protein 2 9 127/565 22.5 3 CSNK2A1 Casein kinase 2, alpha 1 polypeptide 9 65/397 16.4 4 IGF2BP1 Insulin-like growth factor 2 mRNA-binding protein 1 9 104/577 18.0 5 PPP2R1B Isoform 1 of Serine/threonine-protein phosphatase 2A 5 72/601 12.0 65 kDa regulatory subunit A beta isoforms. 6 PTPN9 Protein tyrosine phosphatase, nonreceptor type 9 6 73/593 12.8 7 CRKL Crk-like protein 12 120/303 39.6 8 TBRG4 TBRG4 cDNA FLJ56153 6 65/642 10.1 9 GNL3 Isoform 2 of Guanine nucleotide-binding protein-like 3 10 124/537 23.1 10 PLK1 Serine/threonine-protein kinase PLK1 6 65/603 10.8 11 SHOC2 Leucine-rich repeat protein SHOC-2 8 91/582 15.6 12 IGF2BP2 Insulin-like growth factor 2 mRNA-binding protein 2 6 73/599 12.2 13 CDKN2AIP CDKN2A interacting protein 8 108/580 18.6 14 METAP2 Methionine aminopeptidase 2 13 123/478 25.7 15 LYRIC MTDH Protein LYRIC 9 128/582 22.0 16 ARCN1 Coatomer subunit delta variant 2 8 84/552 15.2 17 GRK6 Isoform GRK6A of G protein-coupled receptor kinase 6 9 109/576 18.9 18 PABPC1 Isoform1 of Polyadenylate-binding protein1 15 161/636 25.3 19 IKIP Isoform 1 of Inhibitor
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