Supporting Information

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1 Supporting Information Zorde Khvalevsky et al..73/pnas mm sikrsg Molecule PG Matrix ± mm right sc left retained in OER E T= days T= days (iver) T= days (PS) Fig. S. OER characteristics. () iagram of the OER matrix. PG, poly(lactic-co-glycolic) acid. () Magnetic resonance imaging (MRI) detects the OER in the liver of mice; arrow points to the three inserted OERs (one lower OER and two above)., liver; sc, spinal cord. () OER visualization using microcomputer tomography (μt). OERs were implanted into xenograft tumors originating from the human pancreatic cancer cell line apan. t 57 d following implantation, mice were killed and tumor tissue was extracted and visualized by μt. OER (in the circle) located within the tumor (black arrow). () sirn retained in OER: OERs were incubated in phosphate buffed saline (PS) (ph = 7.) or mouse liver tissue ex vivo at 37. was extracted from OERs at different time points and the amount of embedded was measured using three different methods: high-performance liquid chromate (HP), gel electrophoresis, and Nanorop (absorption). (E) OERs protect from degradation: protection of OER-embedded from degradation was assessed by HP. Presented are HP graphs showing : T = d, pure ; T = d (liver), extracted from OER after d incubation in liver at 37 (ex vivo); T = d (PS), extracted from OERs after d incubation in PS at 37. Zorde Khvalevsky et al. of5

2 bp bp mock h h ntc Fig. S. RE: site-specific, -directed cleavage of KRS message. Panc cells were transfected with and harvested after and h. cn was made using KRS-specific primer. 5 poly- tail was added with terminal transferase and dtp. PR amplification of the RE-specific target was done using 3 KRS-specific primer and an oligo-dt primer containing bp at the 3 complementary to the 5 nick site. The results show specific amplification of the treated samples and a time-dependent activity with a peak at h posttransfection (Upper). Sequencing results confirm cleavage at a specific site (ower). relative KRS /β-actin protein level 5 3 KRS protein level in Panc cells OER transfection OER transfection O 8 uciferase level: Panc-luc siuc siuc 8 hrs 7 hrs 7 hrs 9 hrs Fig. S3. The silencing effect of the OER in human pancreatic cancer cells. () Panc-uc cells were incubated with OERs containing either or in the presence of ipofectamine or transiently transfected with these sirns using ipofectamine. fter 8 h, cells were lysed and total level of KRS protein was assessed by Western blot analysis. Representative quantification results (TIN software) of the protein levels (normalized to the level of β-actin) of four independent experiments (± SEM) are shown. () Panc-uc cells were incubated with OERs containing or siuc or in the presence of ipofectamine. fter 7 and 9 h, the cells were harvested and the luciferase level was assessed. The representative result of four different samples is shown. The results are shown ± SEM; P value was calculated relative to of the corresponding time point. P <.5; P <. according to the Student t test. Zorde Khvalevsky et al. of5

3 migrated cells, % of scrambled % % 8% % inhibits Panc migration p<. scrambled TGFb wound width (rela ve).8... inhibits Panc migra on 5 Hours 5 5 NT Scrambled G Fig. S. reduces epithelial mesenchymal transition in Panc cells. () reduces the migration ability of Panc cells. Panc cells were transfected with, nontargeting control (scrambled), or treated with TGFβ (positive control). The migration ability was assessed using a modified oyden chamber assay. The presented percentage of migrated cells relative to untreated cells is presented. () Scratch assay: Panc cells were transfected with or nontargeting control (scrambled). The next day, cells were counted and reseeded to 9% confluency and the scratch assay was performed. The graph represents average wound width. average luciferase level x 3 uciferase level in mice livers of MUP-uc Tg mice expressing uciferase in the livers ays 9 ays luciferase activity X 5 3 uciferase activity in OER-treated mice OERs siuc OERs empty OER siuc OER ay ay 3 Tumor weight days after OER treatement 8 mg OERs siuc OERs Fig. S5. The in vivo silencing effects of siuc OERs. () uciferase activity in the livers of mice that stably express luciferase (uc) in the liver after implantation of an empty or siuc OERs. () uciferase activity in mice bearing s.c. syngeneic T-uc tumors after intratumoral implantation of two siuc or OERs. ()Tumor weight in the group treated with the siuc OERs in comparison with the OERs group. P <.5 according to the Student t test. Zorde Khvalevsky et al. 3of5

4 percentage of necro c area % 5% % 3% Percentage of necro c area in subcutan apan tumors untreated empty OER OER sigv OER siuc OERs volume mm^3 8 OERs Volume of Panc tumors treated with noted OERs 7 days after treatment siuc OER OER E % of survival mice % 8% % % % Survival curve: subcutaneous Panc tumors u/t injected OER empty OER % days after treatment Fig. S. Silencing of KRS inhibited s.c. tumor growth of human pancreatic cancer cells. ( and ) 7 immunostaining of s.c. apan- tumors from the different treatment groups: untreated or implanted with empty OERs, OER, or sigv OER. () Representative slide pictures; arrows point to the OERs location. () alculation of percentage of necrotic area. The calculation was done using ImageJ program. () Tumor volume of Panc s.c. tumors after implantation of siucs or OERs. () Representative slide pictures of Panc s.c. tumors d after implantation of siuc- or OERs., OER location. (E) Survival curve of mice bearing Panc s.c. tumors from the different treatment groups: untreated, intraperitoneally injected with, or implanted with empty OERs or OERs P <.5 according to the Student t test. Zorde Khvalevsky et al. of5

5 percentage of viable cells % 9% 8% 7% % 5% % Viability test Panc Panc Fig. S7. The silencing effect of the in pancreatic cancer cells. Panc and PancO cells were transfected with or nontargeting control,, using ipofectamine. fter 7 h, cell viability was assessed by methylene blue test. Shown is average percentage of viable cells (± SEM). P <.5. Fig. S8. sigv OERs inhibit growth of pancreatic tumors of human origin. Survival curve in mice bearing apan pancreatic tumors from the different treatment groups: implanted with sigv, empty, or OERs intraperitoneally (i.p.) injected with PS, sigv, or. T 7 5 pos/neg K-RS IH staining in pancrea c Panc tumors posi ve/nega ve K-RS staining, as a func on of distance from OER Expon. (pos/neg) pos/ neg distance from OER [μm] T 7 positive staining, as a function of a distance from OER Fig. S9. OER inhibits growth of pancreatic tumors of human origin. Immunostaining (IH) was performed on pancreatic tumors of human origin treated with the OER, d posttratment. () Representative pictures of KRS IH. Shown squares of.. mm at growing distances from OER are used for calculations. () Graph represents ratio of KRS positively stained vs. unstained cells as a function of distance from OER. () 7 IH., OER place; T, tumor tissue. () Graphs represent percentage of 7 stained vs. unstained cells as a function of distance from OER. Zorde Khvalevsky et al. 5of5