Supplementary Figure Legends

Supplementary Figure S1. Jagged2-ablation using another shRNA exhibited similar metastatic defects.

(A)BxPC-3 cells expressing Jagged2 shRNA exhibited lower Jagged2 expression and inhibited Notch activation. (B) Jagged2 depletion had no effect on cell proliferation. Cell viabilitiesof Jagged2-depleted or control BxPC-3 cells were determined by MTT assays.(C)Jagged2 depletion impeded cell migration in BxPC-3 cells. The wound healing in the presence or absence of Jagged2 was determined by measuring the width of gaps, images of migration (left) and quantification of cell migration index (right) shown on the right. All experiments were performed the same way as in Fig. 2.

Supplementary Figure S2.Jagged2-depleted MIA PaCa-2 cells exhibited metastatic defects.

(A)MIA PaCa-2 cells expressing Jagged2 shRNA exhibited lower Jagged2 expression and inhibited Notch activation. Cells were transfected with scramble or Jagged2 shRNA, total RNA and protein extract were used to analyze Jagged2 mRNA abundance and ICN1. -Actin, labeled as Actin, serves as an internal control. (B) Jagged2 depletion had no effect on cell proliferation. Cell viability was determined by MTT assays. Jagged2 depletion had profound effects on cell migration in MIA PaCa cells. The wound healing(C)and transwell assays (D) in the presence or absence of Jagged2 was determined by measuring the width of gaps, images of migration (left) and quantification of cell migration index (right) shown on the right. Jagged2 depletion down-regulated the expression of EMT-relevant markers (E), key transcriptional factors and tumor metastatic factors (F). Gene expression was analyzed by real-time PCR or western blot analysis.All experiments were performed the same way as in Fig. 2.

Supplementary Figure S3. Jagged1 knockdown had no effect onMIA PaCa-2 cells migration.

(A)BxPC-3 cells expressing two distinct Jagged1 shRNAs exhibited lower Jagged1 expression and inhibited Notch activation. Cells were infected with lentiviruses expressing scramble or Jagged2 shRNA, total RNA and protein extract were used to analyze Jagged2 mRNA abundance and ICN1. -Actin, labeled as Actin, serves as an internal control. (B) Jagged1 depletion had no effect on cell proliferation. Cell viability was determined by MTT assays.(C)Jagged1 depletion had no effect on BxPC-3 cell migration, assayed by wound healing for24 hours.Images of migration (left) and quantification of cell migration index (right) shown on the right. Data were presented as the mean values (± SD) of replicate (triplicate) wells.

Supplementary Figure S4.-secretaseinhibition did not impairMIA PaCa-2 cell migration.

(A)Effect of GSI on MIA PaCa-2 cell proliferation. Cells were treated with two types of GSI, Compound E (5 M) orDAPT (5 M), as well as DMSO for 48 hours.(B)Cell viability at each time point was determined by MTT assays. Data were presented from triplicate wells ± SD.(C) Effects of GSI on cell migration in wound-healing assays.Cells were serum starved for 24 hours, treated with Compound E (5M) and DAPT (5M) for 24 or 48 hours. Represented photographs and migration index were shown.

Supplementary Figure S5. Jagged1 and Jagged2-Fc are capable of inducing Notch signaling pathway. To validate the quality of Jagged1 and Jagged2-Fc, 50 g/mL IgG, Jagged1 or Jagged2-Fc were added into BxPC-3 cell culture for 48 hours, followed by assessment of Hes1 expression that reflects Notch activity.