Supplementary Figure 1. MiR-30a binding sites in NOTCH1 and NOTCH2 3’UTRs. The miR-30a binding site in NOTCH1 (nucleotides 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 and NOTCH2.2 at nucleotides 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.

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.

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)

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 miR- 30c 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 t- test), ns = not significant. Relative miRNA-30 expression data represent the mean ± SD of two independent biological replicates, each performed in duplicate (four data points).

Supplementary Figure 5. MiR-30a regulates grow rate of T-ALL cell lines. A) The growth pattern of two NOTCH-1 mutant T- ALL 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 t- test). 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. A) B)

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

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.