Delivery of Therapeutics Targeting the mRNA-Binding Protein HuR Using 3DNA Nanocarriers Suppresses Ovarian Tumor Growth

Supplementary Data

• Supplementary Figure S1 - Supplementary Fig. S1. Location of HuR (human) inhibitory oligonucleotides.
• Supplementary Figure S2 - Supplementary Fig. S2. A. HuR mRNA levels measured by RT-PCR in A2780 transiently-transfected with siHuR and siCtrl. B. Constitutively expressed shHuR maintains inhibition of HuR expression in ovarian tumor cells. Western blots of total cell lysates of parental and shHuR-constitutively expressing A2780, OVCAR5, and OVCA420 cells at various passage numbers. C. HuR mRNA levels measured by RT-PCR in late passages of A2780 and OVCAR5 cells expressing shHuRc or shCtrl. * = p<0.0005
• Supplementary Figure S3 - Supplementary Fig. S3. A. HuR mRNA levels in parental and transfected OVCAR3 cells upon shHuR induction with 1mg/ml DOX. PAR = parental. B. Western blots of cell lysates following incubation with various concentrations of doxycycline. C. OVCAR3 toxicity following incubation with various concentrations of doxycycline.
• Supplementary Figure S4 - Supplementary Fig. S4. TUNEL and Ki67 staining for detection of proliferating and apoptotic cells, respectively. A. TUNEL and Ki67 stained sections of four OVCAR5-shHuR and four OVCAR5-shCtrl ovarian xenografts. Areas of Ki67- cells are delineated with a white dotted line. B. In vitro staining of parental OVCAR5 cells, OVCAR5-shCtrl cells and OVCAR5-shHuR cells. Ki67+ cells and DAPI-stained cells were counted in three 1.4mm2 fields to determine % Ki67+ cells. * indicates p = 0.003 between parental and shHuR cells. DAPI stains nuclei in panels A and B. White bar = 50 mm.
• Supplementary Figure S5 - Supplementary Fig. S5. A. 3DNA^® dendrimer platform for in vivo tumor-targeted delivery of siHuR. B. Ovarian cancer cell lines, non-tumorigenic human immortalized ovarian epithelial cells (HIO 80 and HIO 120), and murine ID8-Fluc cells express the folate receptor. C. Serum stability. siHuR incubated for 1 hr at 37^oC, then assayed on a 10%Tris/Borate/Urea PAGE. Sense strand: 42 bases (modified bases + extension); Antisense strand: 22 bases (modified bases). D. In vitro siHuR knockdown activity. siHuR (13nM) was combined with lipofectamine or targeted 3DNA dendrimer and incubated with A2780 cells overnight in 10% serum-containing medium. Dendrimer plus a scrambled siHuR had no knockdown activity (not shown).
• Supplementary Figure S6 - Supplementary Fig. S6. Biodistribution of Cy3-conjugated 3DNA +/- conjugation to folic acid (FA). Following systemic delivery of FA-targeted Cy3-3DNA to ovarian tumor bearing mice, significant Cy3 fluorescence was observed in ovarian tumors, lesser amount in normal ovaries of tumor-bearing mice, very low amounts in brain, liver, spleen, and lung (white arrows), and no fluorescence was observed in heart. No fluorescence was observed in the ovary of non-tumor bearing mouse.
• Supplementary Figure S7 - Supplementary Fig. S7. A. Principal component analysis (PCA), and B. Hierarchical Clustering (HCL) Analysis showing distinct difference between transcript profile of siCtrl- and siHuR-transfected A2780 cells. C and D. MA plots of gene expression of 163 genes following transfection of A2780 cells with siHuR and with siCtrl. C. 66 genes were significantly down-regulated and 50 genes were significantly up-regulated when HuR was knocked down. D. 37 significantly down-regulated genes (56%) are direct HuR targets (i.e., HuR binds to their mRNA transcript); 31 significantly up-regulated genes (62%) are direct HuR targets.
• Supplementary Table S1 - Supplementary Table S1. Biological function of 5 up-regulated and 6 down-regulated genes having the highest fold differences in expression in response to HuR inhibition in A2780 cells. Shaded boxes indicate transcripts to which HuR binds directly.