Phenotypic Screening Identifies Protein Synthesis Inhibitors As H- Ras-Nanocluster Increasing Tumor Growth Inducers

Supporting Information for: Phenotypic Screening Identifies Protein Synthesis Inhibitors as H- Ras-Nanocluster Increasing Tumor Growth Inducers Arafath K. Najumudeen1, Itziar M. D. Posada1, Benoit Lectez1, Yong Zhou2, Sebastian K. -J. Landor1, 3, Adyary Fallarero4, Pia Vuorela4, John Hancock2, Daniel Abankwa*1 Author affiliations 1 Turku Centre for Biotechnology, Åbo Akademi University, Tykistökatu 6B, 20520 Turku, Finland 2 University of Texas Health Science Center at Houston, Medical School, Houston, Texas 77030, United States 3 Department of Cell and Molecular Biology (CMB), Karolinska Institutet, SE- 171 77 Stockholm, Sweden 4 Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, FI-00014 Helsinki, Finland * e-mail: [email protected] Page 1 of 23 Supporting experimental procedures β -galactosidase enzymatic assay BHK and HEK cells were seeded on 12-well plates and transfected with pCMV-β– Gal plasmid at 150 or 50 ng/mL, respectively, using jetPRIME reagent. A negative control sample was included (untransfected) in order to follow the endogenous β–gal activity of the cell. Cells were subsequently incubated with the compounds for 16 h at the following concentrations: CHX at 0.18 or 10 µM, anisomycin and harringtonine at 10 µM. The cells were next lysed with PBS buffer containing 1% Triton-X-100 and 100 µg/mL PMSF and samples were centrifuged (15,000 rpm, 5 min, 4 °C) to remove cell debris. 10-to-20 µl of cell lysate was then incubated with 100 µl reaction buffer containing ortho-nitrophenyl-β-galactoside (0.6 mg/mL ONPG in 0.1 M sodium phosphate buffer, 1 mM MgCl2 and 45 mM β-mercaptoethanol) and β–galactosidase activity was followed spectrophotometrically at λ= 420 nm on a Synergy™ H1 Multi- Mode Reader (BioTek, VT, USA). For β–gal activity normalization, the absorbance was normalized with protein amount, measured by the bicinchoninic acid assay (Thermo-Fischer Scientific). Western Blot For the protein synthesis inhibition assay (Supporting Information Figure 2a), BHK cells were treated for 24 h with CHX at 0.18 or 10 µM, and anisomycin and harringtonine at 10 µM. Cell lysates were run on an SDS-PAGE and electroblotted, and membranes were immunolabeled with anti-p53 antibody (Cell Signaling Technology, cat. no. 9282) or β-Actin which was used as loading control, and membranes were developed as described on methods section. For the signaling assay (Supporting Information Figure 3b) BHK cells were treated with anisomycin and harringtonine at 2 µM for 24 h. FLIM-FRET For the FLIM experiments on Supporting Information Figure 3a, HEK cells were seeded on a 6-well plate with glass coverslips, and transfection was done after 24 h using jetPRIME (Polyplus) transfection reagent with pmGFP-H-rasG12V alone or together with pmCherry-H-rasG12V (pmGFP/pmCherry plasmids at 1:3 ratio, 2 µg total plasmid). 24 h after transfections, cells were treated for 24 h with either control Page 2 of 23 (DMSO 0.05% (v/v)) or 0.18 µM of CHX or 2 µM of anisomycin or harringtonine and incubated for additional 24 h. Cells were fixed in 4% PFA/PBS for 20 min and then washed in PBS, and coverslips with cells were then mounted with Mowiol 4-88 (Sigma Aldrich, cat. no. 81381) on glass slides. Cell proliferation assays For MDA-MB-231 cell proliferation assay, cells were seeded in 96-well plates and treated after 24 h with either control (DMSO 0.05% (v/v)) or 10 µM CHX and incubated for 72 h. DMSO concentration in the final samples was kept under 0.05% (v/v). Cell proliferation was measured every 24 h as change in fluorescence intensity using alamarBlue (Invitrogen, #DAL1100). Each treatment condition comprised of at least five replicate wells and was repeated three times. The fluorescence intensities were expressed as change in cell proliferation relative to the control. Data analysis was performed using GraphPad Prism. Page 3 of 23 a 0.6 positive deviators R2 = 0.87 0.55 Anisomycin CHX 0.5 Harringtonine Emetine 2HCl (HEK) control Actinomycin D max 0.50 0.4 Echinomycin Diacetoxyscirpenol 0.3 FRET E FRET 0.45 0.2 control 0.2 0.3 0.4 0.5 0.6 0.5 0.6 FRET Emax (BHK) FRET Emax (BHK) negative deviators control 0.4 (HEK) max Tubercidin 0.3 Lapachone, b DHTS Mevastatin FRET E FRET A-23187 2-Hydroxy acetophenone 0.2 0.2 0.3 0.4 0.5 FRET E (BHK) max Supporting Information Figure 1. Cross-validation of hits in HEK cells. (a) Correlation analysis between Emax values from HEK and BHK cells expressing H- ras-NANOPS. Cells were treated for 24 h with 10 µM of hits from the screen. Dots represent average Emax in HEK and BHK cells from three independent experiments. Block lines indicate the line fit with 95% confidence interval band. Dotted lines mark the hit selection thresholds; red - increase in Emax and blue - decrease in Emax from the average of the control (green). Right, zoomed from (a) showing the positive deviators marked in red. Left, zoomed from (a) showing the negative deviators marked in blue. Page 4 of 23 M M μ a μ M μ M μ controlCHX 0.18CHX 10Anisomycin Harringtonine 10 10 1 0.12 0 0 0 p53 β-actin b c 100 ) ns ) 100 **** 50 **** 50 -gal activity (% -gal activity (% 0 0 l d M M M M d l µ µ µ µ ro M M M M µ µ µ µ 10 ont sfectecontro 10 c 10 10 n X in X in tra CH CH n CHX 0.18 myc myc u o untransfecte CHX 0.18 o s s ni ni A A Harringtonine 10 Harringtonine 10 Supporting Information Figure 2. PSI treatment inhibits protein translation. (a) p53 (t1/2= 10 min) expression level was measured under PSI treatment in BHK cells. Cells were treated with the compounds for 24 h and cell lysates were immunoblotted against p53 and β-Actin (n=1). (b, c) β-galactosidase enzymatic activity was measured under PSI treatment. BHK cells (b) and HEK cells (c) were transfected with pCMV-β-gal and subsequently incubated with the following compounds for 16 h: CHX (at 0.18 and 10 µM) anisomycin (10 µM) and harringtonine (10 µM). Cell lysates were incubated with ONPG and β-gal activity was followed spectrophotometrically (mean ± SEM, n≥2). Loss of β-gal activity was related to loss of protein translation. Statistical significance between control and treated sample was determined using one-way ANOVA test (ns, not significant; ****, p<0.0001). Page 5 of 23 a b control AnisomycinHarringtonine control 246 1 7.3* 8.7* + Anisomycin 2 μM 112 ppErk1/2 + Harringtonine 2 μM 139 **** Erk1/2 + CHX 0.18 μM 171 1 2.8* 2.5* 10 15 20 25 pAkt apparent FRET efficiency (%) Akt β-actin Supporting Information Figure 3. PSIs increase H-ras nanoclustering and Erk- Akt signaling. (a) Nanoclustering-FRET analysis in HEK cells co-expressing mGFP- and mCherry- tagged H-rasG12V. Cells were treated for 24 hours with either DMSO control or the indicated protein synthesis inhibitors. The apparent FRET efficiency (mean ± SEM, n=3) was calculated from FLIM data. Statistical significance between control and treated cells were examined using one-way ANOVA tests (****, p<0.0001). The numbers in the bars indicate the number of analyzed cells. (b) Representative western blots for BHK cells treated for 24 h with either DMSO control or 2 µM anisomycin and harringtonine. Numbers indicate the ratio of the phosphorylated Erk and Akt relative to total Erk and Akt, respectively. β-Actin was used as a loading control (mean ± SEM, n=3). Statistical significance was determined using Student’s t-test (*, p<0.05). Page 6 of 23 a 6 ) control CHX 10 μM 4 2 cell proliferation (relative to control 0 0 24 48 72 time (h) Supporting Information Figure 4: CHX inhibits normal proliferation of MDA- MB-231. Proliferation of MDA-MB-231 cells treated for 72 h with either DMSO or CHX at 10 µM. The graph shows mean fold change compared to DMSO control at each time point ± SEM (n=6). Page 7 of 23 Supporting Information Table 1: List of compounds included in the commercially available Enzo® Screen-Well® Natural Product Library (Enzo Life Sciences Inc., USA) and the in-house diverse natural compounds collection obtained from different commercial suppliers. ID codes of the compounds for each library are indicated with their corresponding molecular weight (MW). ENZO codes Compound Name MW 1 Acivicin 178.6 2 Actinomycin D 1255.5 3 Anisomycin 265.3 4 Antibiotic A-23187 523.6 5 Aristolochic acid A 341.3 6 Artesunate 384.4 7 Australine HCl 225.5 8 Baicalein 270.2 9 Betulinic acid 456.7 10 Bilobalide 326.3 11 Brefeldin A 280.4 12 Bromocriptine mesylate 750.7 13 C2 Phytoceramide 359.6 14 C6 Ceramide 397.7 15 Caffeic Acid 180.2 16 Camptothecin 348.4 17 Cantharidin 196.2 18 CAPE 284.3 19 Capsaicin 305.4 20 Castanospermine 189.2 21 Cerulenin 223.3 22 Cevadine 591.7 23 Chaetomellic acid A 370.4 24 Chelerythrine Cl 383.8 25 Chromomycin A3 1183.3 26 Citrinin 250.3 27 Colchicine 399.4 28 Coumermycin A1 1110.1 29 Curcumin 368.4 30 Cycloheximide 281.1 31 Cyclopamine 411.6 32 Cyclopiazonic acid 336.4 33 Cycloserine, L- 102.1 34 Cyclosporin A 1202.6 35 Cytochalasin B 479.6 36 Cytochalasin D 507.6 37 Cytochalasin E 495.6 38 Daidzein 254.2 39 Daunorubicin HCl 564 40 Decoyinine 279.2 41 Deguelin 394.3 42 Deoxyphorbol 13-acetate, 12- 390.5 43 Deoxyphorbol 13-phenylacetate 20-acetate, 12- 508.6 44 Dihydroergocristine mesylate 707.8 45 Domoic acid 311.3 46 Doxorubicin HCl 580 47 E6 Berbamine 757.8 48 E-64 357.4 49 E-64-C 314.4 50 E-64-D 342.4 Page 8 of 23 51 Ebelactone B 352.5 52 Ellipticine 246.3 53 Embelin 294.4 54 Epibatidine, (±)- 208.7 55 Epigallocatechin gallate 458.4 56 Etoposide 588.6 57 Forskolin 410.5 58 Fumagillin 458.6 59 Fumonisin B2 705.8 60 Galanthamine HBr 368.3 61 Gambogic acid 628.8 62 Genistein 270.2 63 Geranylgeranoic acid 304.5

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