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Supplementary Figures an Immature Subset of Neuroblastoma Cells 1 Supplementary Figures 2 3 An immature subset of neuroblastoma cells synthesizes retinoic acid and 4 depends on this metabolite 5 6 7 Tim van Groningen, Camilla U. Niklasson, et al. 8 1 Supplementary Figure 1 a control 1 µM RA 5 µM RA 10 µM RA S - 26% S - 24,9% S - 19,9% S - 19,1% 691-MES S - 47,1% S - 28,3% S - 21,8% S - 22,5% 691-ADRN EdU Propidium Iodide b control 1 µM RA 5 µM RA 10 µM RA S –21,2% S -15,9% S -11% S -11% 717-MES S - 28,3% S - 10% S - 6,1% S - 7,4% 717-ADRN EdU Propidium Iodide 9 Supplementary Figure 1 – related to Figure 1. 10 Proliferation of MES and ADRN cells in response to RA. 11 A, B. EdU incorporation assay and Propidium-Iodide (PI)-staining for MES and ADRN cells 12 from patient 691 (in A) and patient 717 (in B). 691-MES, 691-ADRN, 717-MES and 717- 13 ADRN cells were treated with 1, 5 and 10 μM RA for 5 days and analyzed by FACS for PI 14 (x-axis) and EdU (y-axis). 15 2 Supplementary Figure 2 a b c MES (n=9) ADRN (n=26) Retinol ALDH1A1 ALDH3B1 500 80 RDH10 kDa 400 - 55 Retinal 60 ALDH1A1 300 ALDH1A3 - 56 ALDH DEAB 40 YAP1 - 70 200 RA + RAR BMS493 GATA3 - 48 ER50089 20 mRNA expression mRNA 100 mRNA expression mRNA Total AKT - 60 RARE-luciferase 0 0 d e g 8 691-MES 691-ADRN 717-MES 717-ADRN 691-MES SH-EP2 691-ADRN 0 120 8 *** *** SH-SY5Y 100 +ROL 0 80 60 40 - ROL Cell number (%) 20 0 0 100 150 20 f SH-EP2 +ROL -ROL -ROL -ROL DEAB (µM) 0 control +100 nM RAL +100 nM RA 20 717-MES SH-SY5Y 717-ADRN 0 120 20 *** *** NBLW-MES 100 0 691-MES 80 20 NBLW-ADRN 60 0 40 20 Cell number (%) 717-MES 0 0 100 150 DEAB (µM) 16 Supplementary Figure 2 – related to Figure 1. 17 MES neuroblastoma cells have an active RA synthesis pathway. 18 A. Schematic overview of the core RA synthesis pathway, according to15,16. Retinol is 19 converted to retinal by RDH10. Retinal is converted to RA by ALDH enzymatic activity, the 20 latter can be inhibited by DEAB. RA associates with Retinoic Acid Receptors (RARα, 21 RARβ, RARγ). The RARα inhibitor ER50891 and the pan-RAR inhibitor BMS493 can block 22 the RA/RAR transcriptional activity. A RARE-luciferase reporter gene responds to an 23 endogenous source of RA. 24 B. mRNA expression of ALDH1A1 (left) and ALDH3B1 (right) genes, measured by 25 Affymetrix profiling in MES (n = 9, in orange) and ADRN (n = 26, in blue) cell lines. 26 C. Western blot analysis of ALDH1A1, ALDH1A3, the MES-marker YAP1 and the ADRN- 27 marker GATA3 in isogenic cell line pairs 691-MES and 691-ADRN, 717-MES and 717-ADRN 28 and SH-EP2 and SH-SY5Y. Cell lines of MES or ADRN phenotype are shown in orange and 29 blue, respectively. Total AKT is shown as loading control. 30 D. ChIP-sequencing analyses of H3K27ac of the genomic regions around the ALDH1A1 or 31 ALDH3B1 genes. The y-axis represents reads per 20 million mapped sequences. The 32 genomic position of the genes on sense (green) or antisense (red) DNA strands is shown 33 on the x-axis. 34 E. Bright field images of 691-MES, 691-ADRN, 717-MES and 717-ADRN cultured in 35 neural stem cell medium with retinol (+ROL, upper panels) or without retinol (-ROL, lower 36 panels) for 14 days. Scale bars represent 50 µm. 37 F. Bright field images of 691-MES (upper panels) and 717-MES (lower panels) cells 38 cultured in the presence (+) or absence (-) of retinol (ROL) for 18 days. At day 14, 100 nM 39 Retinal (RAL) or 100 nM RA was added as indicated. DMSO was added as control. Scale 40 bar represents 50 µm. 41 G. CyQuant cell viability assay of isogenic cell-line pairs 691-MES/-ADRN (left) and 717- 42 MES/-ADRN (right) with increasing concentrations of the ALDH-inhibitor DEAB. MES cell- 43 lines are shown in orange and ADRN cell-lines in blue. Two-sided Student’s t-test assuming 3 44 equal variance was used to calculate statistical significance, *** p < 0.001. Source data are 45 provided as a Source Data File. 46 4 Supplementary Figure 3 a b e 691-MES 691-ADRN 717-MES 717-ADRN 80 7 7 7 - DEAB 72.3 +/ 4.1% 7 - DEAB - DEAB - DEAB 10 10 10 - 10 + 13.0 / 1.7% + + 60 - 72.8 / 2.4% 31.0 /- 3.4% 6 6 6 6 - 10 10 10 10 40 5 5 5 5 SSC-A SSC-A 10 20 10 10 10 Aldefluor+ (%) Aldefluor+ 4 4 4 4 0 10 10 10 10 2 10 3 10 4 10 5 10 6 10 7 1010 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 1 2 3 4 7 7 7 + DEAB 0.1% 7 + DEAB + DEAB + DEAB 10 10 10 10 0.1% 0.1% 0.1% 6 6 6 6 10 10 10 10 5 5 5 5 SSC-A SSC-A 10 10 10 10 4 4 4 4 10 10 10 10 2 10 3 10 4 10 5 10 6 10 7 1010 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 Aldefluor signal Aldefluor signal Aldefluor signal Aldefluor signal c d SH-EP2 SH-SY5Y NBLW-MES NBLW-ADRN 7 7 7 - DEAB 7 - DEAB - DEAB - DEAB 10 10 10 + 10 44.3 /- 5.0% + + + 5.6 / 4.1% 34.5 / 5.4% 3.5 /- 1.3% 6 6 6 6 - - 10 10 10 10 5 5 5 5 SSC-A SSC-A 10 10 10 10 4 4 4 4 10 10 10 10 2 10 3 10 4 10 5 10 6 10 7 1010 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 7 7 7 + DEAB 7 + DEAB + DEAB + DEAB 10 10 10 0.1% 10 0.1% 0.1% 0.1% 6 6 6 6 10 10 10 10 5 5 5 5 SSC-A SSC-A 10 10 10 10 4 4 4 4 10 10 10 10 2 10 3 10 4 10 5 10 6 10 7 1010 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 10 2 10 3 10 4 10 5 10 6 10 7 Aldefluor signal Aldefluor signal Aldefluor signal Aldefluor signal 47 Supplementary Figure 3 – related to Figure 1. 48 ALDH activity marks MES-type neuroblastoma cells. 49 A-D. Flow cytometry analysis of ALDH activity measured by Aldefluor-assay in four isogenic 50 MES and ADRN cell line pairs (A) 691-MES/691-ADRN, (B) 717-MES/717-ADRN, (C) SH- 51 EP2/SH-SY5Y and (D) NBLW-MES/NBLW-ADRN in the absence (-, upper panel) or 52 presence (+, lower panel) of the ALDH-inhibitor DEAB. The gate includes Aldefluorpositive cells 53 sensitive to DEAB-treatment. A representative FACS analysis from three independent 54 Aldefluor experiments is shown. The percentages of Aldefluorpositive cells are averages of 55 three independent measurements +/- standard deviation. Aldelfuor signal is shown on the x- 56 axis, side-scatter (SSC-A) is shown on the y-axis. 57 E. Summary of Aldefluorpositive cells in the four isogenic MES and ADRN cell line pairs. Shown 58 are the average measurements of three independent FACS experiments. In each 59 experiment, 10,000-20,000 cells were analysed. The error bar indicates standard deviation. 60 MES cell lines are shown in orange, ADRN cell lines are shown in blue. The percentage of 61 Aldefluorpositive cells is shown on the y-axis. Source data are provided as a Source Data File. 62 5 Supplementary Figure 4 a c 691-MES 717-MES 691-MES 717-MES 300 *** 250 *** 120 120 250 200 100 100 200 80 80 150 *** * ** ** 150 *** 60 60 100 100 40 40 *** *** *** 50 50 20 20 RARE-luciferase activity RARE-luciferase activity RARE-luciferase activity 0 0 0 RARE-luciferase activity 0 - - - - + + ER50891 control + + ER50891 control-- + + RA -- + + RA 0 0.1 1 10 0 0.1 1 10 µM ER50891 µM ER50891 b d 691-MES 717-MES 691-MES 717-MES 300 250 *** 120 120 250 100 100 *** 200 200 80 80 150 150 *** *** 60 60 100 100 40 40 50 50 20 *** *** *** 20 *** *** *** RARE-luciferase activity RARE-luciferase activity RARE-luciferase activity 0 0 0 RARE-luciferase activity 0 - + - + BMS493 - + - + BMS493 control-- +RA+ + RA control-- +RA+ + RA 0 0.1 1 10 0 0.1 1 10 µM BMS493 µM BMS493 e f 691-MES 717-MES shALDH1A3 shALDH1A3 140 140 691-MES 717-MES * ** shALDH1A3 shALDH1A3 120 120 100 100 - + IPTG - + IPTG 80 80 - 56 - 56 ALDH1A3 ALDH1A3 60 60 β-actin - 45 β-actin - 45 40 40 % RARE-luciferase … % RARE-luciferase % RARE-luciferase … % RARE-luciferase 20 20 RARE-luciferase activity 0 RARE-luciferase activity 0 - + IPTG - + IPTG 63 Supplementary Figure 4 – related to Figure 1.
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