1500.......1510........1520.................1530........ AAAAAUGUAGUUUACA NOTCH1 rat NOTCH1 mouse UAAAGUGUAGUUUACA NOTCH1 chimpanzee AUAAAAUAGAGUGUAGUUUACA NOTCH1 human 5’ AUAAAAUAGAGUGUAGUUUACAG...3’ ||||||| miR-30a 3' GAAGGUCAGCUCCUACAAAUGU 5’ 240........250.......260.......270........280...........290.. UUGGGGCCCC--UCGCU NOTCH2.1 rat UUGAGACCCC-UUGCUC NOTCH2.1 mouse CAAGCCUUGGGUCCAUGUUUACU NOTCH2.1 chimpanzee 5’ CAAGCCUUGGGUCCAUGUUUACU. . .3’ NOTCH2.1 human ||||||| 3' GAAGGUCAGCUCCUA CAAAUGU 5’ miR-30a ......1980......1990..........2000................2010... AUGGCAGU--------NOTCH2.2 rat ACAGCAGU-CUA---UA NOTCH2.2 mouse NOTCH2.2 chimpanzee GUUGGCAUAAUAGUUUACAAAU 5’ GUUGGCAUAAUAGUUUACAAAU 3' NOTCH2.2 human ||||||| 3' GAAGGUCAGCUCCUACAAAUGU 5’ miR-30a Supplementary Figure 1. MiR-30a binding sites in NOTCH1 and NOTCH2 3’UTRs. The miR-30a binding site in NOTCH1 (nucleotides 1520-1526 of the human 3’UTR sequence) is highly conserved (top panel), whereas the two NOTCH2 binding sites (middle and bottom panels) are present only in primates (NOTCH2.1 at nucleotides 257-263 and NOTCH2.2 at nucleotides 19911997 of the human 3’UTR sequence). In addition to seed sequence (red font), all three sites show an additional complementary nucleotide – seed match 7mer-1A for NOTCH1; 7mer-m8 and 7mer-1A for NOTCH2.1 and NOTCH2.2, respectively. 5 KOPTK1 miR-30a KOPTK1 MSCV DND41 miR-30a 0 DND41 MSCV KOPTK1 miR-30a KOPTK1 MSCV DND41 miR-30a DND41 MSCV DHL7 miR-30a DHL7 MSCV DHL6 miR-30a DHL6 MSCV 0 10 DHL7 miR-30a 2 15 DHL7 MSCV 4 20 DHL6 miR-30a 6 25 DHL6 MSCV miR-30a relative expression miR30a Delta CT values 8 Supplementary Figure 2. Expression of miR-30a in genetically modified DLBCL and T-ALL cell lines. Stable integration of MSCV-30a constructs elevated the expression of this miRNA in all four cell line models created. In the left panel, the data are displayed as Delta CT (miR-30a values normalized by control snoRNA U18 values) and, as expected, in all instances the DCT value is lower (i.e., higher gene expression) for miR-30a expressing cells than for the isogenic controls containing an empty MSCV-eGFP construct. In the right panel, we display the relative expression of miR-30a in each cell line, as they compare to their isogenic MSCV control. All miRNA and snoRNA quantifications were performed with the stem-loop RT-PCR assay; data show are mean ± SD of triplicates. Sponge control Sponge miR-30a Luciferase activity 500000 P<0.05 400000 300000 200000 100000 0 NOTCH1 WT NOTCH1 Mut Supplementary Figure 3. Activity and specificity of miR-30a-directed sponge constructs. HEK-293 cells stably expressing the miR-30a-directed sponge or the inert control sponge constructs were transiently transfected with the pmiR luciferase-NOTCH1 binding site reporter plasmid (WT or mutant), miR-30a synthetic oligonucleotides and the pCMVβ-gal plasmid (for normalization purposes). Synthetic miR-30a oligos were significantly more effective in suppressing the luciferase activity of the NOTCH1-WT construct in cells constitutively expressing an inert control sponge than in those expressing the miR-30a-directed sponge (bars on the left). This result demonstrates activity, as the miR-30a sponge effectively binds to this mature miRNA, sequesters it away from its target, thus limiting luciferase inhibition. Conversely, the luciferase activity of a NOTCH1 binding site mutant reporter was indistinguishable in the cells expressing an inert control or the miR-30a directed sponge, demonstrating specificity. Data shown are mean and ±SD of an assay performed in triplicate. Two biological replicates were completed for this assay. P <0.05 (two tailed Student’s t test) ns 1.0 0.5 * -30e P<0.05 ns -30d P<0.05 * -30c ns * 1.5 1.0 0.5 -30b -30c -30d -30e 24h 48h -30b -30e -30d -30c -30b -30e -30d 0.0 -30c 0.0 -30b miR-30 (relative expression) 1.5 DMSO GSI 2.0 miR-30 (relative expression) MSCV ICN1 72h Supplementary Figure 4. MiR-30 family expression following genetic and pharmacological modulation of NOTCH1. Left panel. Stable expression of intracellular NOTCH1 (ICN1) in the DLBCL cell line SU-DHL7 led to significant downregulation of miR30c and 30d (p<0.05, two-tailed Student’s t-test). Right panel. Exposure of the T-ALL cell line KOPT- K1 to the gamma-secretase inhibitor Compound E (100nM), resulted in significant de-repression of miR-30b, -30c, and 30e; *(p<0.05, two-tailed Student’s ttest), ns = not significant. Relative miRNA-30 expression data represent the mean ± SD of two independent biological replicates, each performed in duplicate (four data points). Cell number (X106) A) MSCV miR-30a 6 Cell number (X106) 10 8 * * 4 * 2 * MSCV miR-30a 8 * 6 4 * * 2 0 0 0 1 2 3 4 0 Days 5 1 2 6 5 Cell number (X10 ) ctrl-sponge miR-30a-sponge 6 Cell number (X106) 6 4 * 3 * * 2 * 1 0 0 1 2 3 DND-41 3 4 Days 5 KOPT-K1 DND-41 B) * 4 5 Days * ctrl-sponge miR-30a-sponge 5 4 * 3 * 2 * 1 0 0 1 2 3 4 5 Days KOPT-K1 Supplementary Figure 5. MiR-30a regulates grow rate of T-ALL cell lines. A) The growth pattern of two NOTCH-1 mutant TALL cell lines stably expressing miR-30a or an empty vector (MSCV) was monitored daily using an automated blue (TB) exclusion assay. In both models, miR-30a expression significantly reduced cell proliferation (upper panels) (* p<0.01, two-tailed Student’s ttest). B) Functional inactivation of miR-30a with stable expression of a specific sponge construct in the same T-ALL cell lines, significantly enhanced their growth rate (lower panels) (p<0.01, two-tailed Student’s t-test). All data are mean ± SD of an assay performed in triplicate. Three biological replicates were performed for each assay, each time in triplicate. 80 P<0.01 P<0.01 NS 2.0 Cells in G0/G1 (%) Apoptosis (fold increase) 2.5 1.5 1.0 0.5 P<0.01 60 40 20 miR30a 13.6% SSC-A Annexin V PE 30 a iR m SC V M 30 a G1: 55.97% S: 17.15% G2M: 22.6% MSCV miR30a G1: 47.23% S: 36.97% G2M: 15.53% G1: 58.00% S: 30.14% G2M: 11.47% MSCV miR30a DND41 miR30a 4.5% SSC-A MSCV KOPT-K1 G1: 54.39% S: 19.89% G2M: 23.30% Annexin V PE Annexin V PE KOPTK1 DND-41 SSC-A DND41 6.10% SSC-A MSCV KOPT-K1 iR M DND-41 m SC V iR m M 0 30 a SC V 30 a iR m M SC V 0.0 9.0% KOPTK1 Annexin V PE Supplementary Figure 6. Cell cycle arrest and/or apoptosis associated with loss of fitness in miR-30a expressing T-ALL cell lines. Expression of miR-30a significantly limited the growth rate of T-ALL cell lines, as determined by daily automated monitoring with a trypan blue exclusion assay (Figure 4A). Cells collected at day 5 of these assays, were analyzed for apoptosis by Annexin V staining (left panel) and their cell cycle profile determined by propidium iodide (PI) staining (right panel). Expression of miR-30a significantly increased apoptotic rate in both models, while G0/G1 arrest was evident in the KOPT-K1. Apoptosis data are represented as mean ±SD of the fold increase in apoptosis in the miR-30a expressing cells compared to MSCV controls. Cell cycle data are displayed as the actual percentage of miR-30a or MSCV controls cells in G0/G1. These assays were all performed in triplicate and three biological replicates completed. NS, non-significant; P <0.01 (two tailed Student’s t test). A representative histogram of each assay/cell line is also shown kD kD SU-DHL7 OCI-Ly18 DND-41 KOPT-K1 34 34 Caspase-3 Caspase-3 26 26 26 26 Cleaved caspase-3 17 Cleaved caspase-3 17 β-actin 30a-spg ctrl-spg 30a-spg ctrl-spg miR-30a MSCV miR-30a MSCV β-actin Supplementary Figure 7. MiR-30a influences apoptosis in T-ALL and DLBCL cell models. Left panel. Ectopic expression of miR-30a in the T-ALL cell lines DND-41 and KOPT-K1 increased the abundance of cleaved caspase3, in association with the expected decrease in intact (inactive) caspase3. These data support the FACS-based Annexin V results shown in Supplementary Figure 6. Right panel. Functional depletion of miR-30a with sponge constructs in the DLBCL cell lines SU-DHL7 and OCI-Ly18, resulted in a decrease in the processing of caspase3, with accumulation of the intact (inactive) enzyme (top) and marked decrease in the cleaved (active) caspase-3 isoform. These data further validate the FACS-based Annexin V results shown in Figure 4D.