Fig 2: Effects of LEN on the Notch signaling pathway in H2O2-stimulated PC12 cells. Relative protein expression of Notch signaling pathway-related Notch2, Hey1 and HES1 in H2O2-stimulated PC12 cells in the presence and absence of LEN at different doses, detected by western blot analysis. *P<0.05 and ***P<0.001 vs. control; ###P<0.001 vs. H2O2. LEN, lenalidomide; Hey1, HES-related family bHLH transcription factor with YRPW motif 1; HES1, hes family bHLH transcription factor 1.

Fig 3: MYC regulates and it is regulated by the miR-30-NOTCH1/2 axisA) Genetic gain of MYC impinges on miR-30a and Notch1 expression. Mature B cells were isolated from the spleens of five Myc Tg mice and four WT littermate controls. MiR-30a levels were significantly lower in B cells expressing the Myc transgene (mean relative expression 0.78±0.1 vs. 1.38±0.3, p<0.05, two-tailed Student’s t-test) and correlated with higher Notch1 expression. Notch2 levels were not distinct among these mice, in agreement with the lack of conservation of the miR-30a binding in murine Notch2. B) Genetic modulation of miR-30a modifies MYC expression in multiple DLBCL and T-ALL cell line models. Western blot analysis depicts suppression or upregulation of MYC upon stable expression of miR-30a (upper panel) or a miR-30a-specific sponge construct (lower panel), respectively.

Fig 4: Genetic and pharmacological modulation of NOTCH1 and NOTCH2 modifies miR-30 expressionA) Stable expression of intracellular NOTCH1 and NOTCH2 (ICN1 and ICN2) in the DLBCL cell line SU-DHL7 led to upregulation of MYC expression, which was accompanied by downregulation of miR-30a (bottom panel) (p<0.05, two-tailed Student’s t-test). MiR-30a relative expression data are mean ± SEM of three independent biological replicates performed in duplicate (six data points) B) Exposure of two T-ALL cell lines to the gamma-secretase inhibitor Compound E (100–200nM), resulted in a temporally related ICN1 suppression (detected as early at 24h), followed by MYC inhibition and subsequently by a significant de-repression of miR-30a (a ~ 50% increase in expression at 72h). (A) MiR-30a relative expression data are mean ± SEM of two independent biological replicates performed in duplicate or triplicate (five data points) *(p<0.05, two-tailed Student’s t-test).

Fig 5: Notch2 inhibition diminishes differentiation-dependent prolactin secretion in HDSC.Cells were incubated with Notch2 blocking antibodies (A) or Notch2 siRNAs (B) and differentiated for 6 days in the presence of cAMP/E2P4. Supernatants were collected and prolactin (PRL) concentrations were measured by ELISA. Normalization to cellular protein concentrations was performed as mentioned in materials and methods. Bars represent mean values ± S.D. of each 3 different experiments performed in duplicates. * indicates p=0.05 compared to non-stimulated control (n.c.); Significant changes (*, p=0.05) between Notch2 ab or si-Notch2-treated cells (open bars) and untreated (black bars) cells are indicated by brackets. IgG ctrl, IgG control; si-ntc, si-non targeting control.