Supplementary Materials s12

+ Sucrose Quil A BP1Mod2 (5 µg) A CD3 T-cells t C o u n

Supplementary Materials

Supplementary Figures

Supplementary Figure S1

A CD3+ T cells

Sucrose Quil A BP1Mod2 (5 µg) t n u o C BP1Mod2 (10 µg) BP1Mod2 (20 µg) BP1Mod2 (40 µg)

CD69 fluorescence (log)

CD69 fluorescence (log) Supplementary Materials

B NK 1.1+ CD3- NK Cells

Sucrose Quil A BP1Mod2 (5 µg)

BP1Mod2 (10 µg) BP1Mod2 (20 µg) BP1Mod2 (40 µg)

CD69 fluorescence (log) Supplementary Materials

Supplementary Figure S1: Impact of different doses of BP1Mod2 siRNA on NK and T cells following I.T. injection of siRNA. Lymphocytes from TDLN of mice treated with liposome-encapsulated BP1Mod2 siRNA were isolated 24 hours post-intratumoral injection. Isotonic sucrose and Quil A were used as negative and positive controls respectively. CD69 expression was analyzed on (A) CD3+ and (B) NK1.1+, CD3- lymphocytes by flow cytometry. Supplementary Materials

Supplementary Figure S2: I.V. delivery of IS-DsiRNAs activates systemic immune response. Mice were injected subcutaneously with 1x106 TC-1 cells and allowed to establish for 10 days. Mice were then injected I.V. with 40µg siRNA, encapsulated in liposomes. After 48hrs spleen were removed and a single cell suspension prepared and stained with fluorescent antibodies and analysed by flow cytometry. Activation of (a) NK cells and (b) T cells were determined. Each plot is pooled from 3 different mice performed in a single experiment. Error bar represents  s.e.m with p<0.001 by *** as determined by ANOVA with Newman-Keuls post test analysis. Supplementary Materials

Supplementary Figure S3: Knockdown of HPV16 E6/E7 can only be observed in vitro, not in vivo. (a) Mice were injected subcutaneously with 1x106 TC-1 cells and allowed to establish for 12-14 days. Tumors were then injected with 10 µg siRNA in Peglyated liposomes and after 24hrs, tumors removed and RNA extracted and used to create cDNA. E7 expression was determined using human k-ras as a normaliser with no group significantly different compared to untreated tumor. (b) TC-1 cells were cultured in silico at a concentration of 1x105 cells/well and transfected with liposome encapsulated siRNAs to a working concentration of 40nM siRNA per well. RNA was extracted, cDNA synthesized and HPV16 E6/E7 expression levels determined via qRT-PCR, with -actin as a housekeeping gene. Data was averaged from two independent experiments, each in triplicate. Unpaired student’s t-test comparing siRNA treated samples to scrambled treated samples is shown (*P <0.5, **P<0.01). Supplementary Materials

Supplementary Figure S4: Transfection with DsiRNAs in vitro induces TC-1 cell senescence and reduces colony forming ability. (a) TC-1 cells were transfected with DsiRNAs, media replaced and left in culture for 10 days before cells were fixed and stained with X-gal solution (pH 6.0) to dye senescent cells blue. The number of senescent cells within an area of 5 field views under 10x magnification was then counted under light microscope using a cell counter. (b) Cells transfected with DsiRNAs were re-plated at a density of 100 cells/well and left in culture for up to 14 days where macroscopic colonies were visible. Cells were then fixed and stained using crystal violet stain and visible colonies were counted using cell counter. Data was averaged from two independent experiments, each performed in triplicate. Error bar represents  s.e.m with p<0.01 by ** and p<0.001 by *** as determined by ANOVA with Newman-Keuls post test analysis. Supplementary Materials

Supplementary Figure S5

Supplementary Figure S5: Schematic representation of siRNA binding sites. The HPV 16 E7 binding sites targeted by IDT-4/IDT-4m7 and IDT-12 are presented as a schematic representation, highlighted in yellow. HPV 16 E7 CTL epitope is underlined and in blue, and part of the HPV16 E6 splice region is presented in green. Nucleotide sequence was obtained from pubmed search, E7 gene ID: 1489079, last accessed September 1st 2013.

Supplementary Table

Primer Sequence Gene E71 F1 5’-CAGCTGGACAAGCAGAACC-3’ E71 R1 5’-TGCCCATTAACAGGTCTTCC-3’ HPV16 E7 β-Actin F 5’-AGCCTCGCCTTTGCCGA-3’ β-Actin R 5’-CTGGTGCCTGGGGCG-3’ Mouse β-Actin Human Ras F1 5’-TGCGTGAGATCCGGCAGCAC-3’ Human Ras R1 5’-GGAGTCCCCCTCACCTGCGT-3’ Human c-Has-ras

Supplementary Table S1: RT-PCR primer sequences.