PMC

Confocal multichannel microscopy of STAT3d and AuNP-NUAP-STAT3d uptake in epithelial cancer cells after incubating with A431 cell culture (A, B) or FaDu cells (C, D); AuNP-NUAP-STAT3d (A - A431 cells; C - FaDu cells); STAT3 duplex (B - A431 cells; D - FaDu cells). Blue- DAPI; Green - anti-EGFR-Alexa Fluor488 for staining cell plasma membrane; Red - Alexa Fluor 568 (one strand of STAT3 duplex).

The uptake of aptamers and aptamer-linked AuNP nanoconstructs in cell culture. A- Total cell uptake of Cy5.5-labeled nucleolin-specific aptamer (NUAP) and control non-specific aptamer (CTAP) incubated with HNC cell line FaDu and squamous carcinoma A431 for 4h. The uptake was determined by using measurements of Cy5.5 fluorescence in cell lysates; B- fractional content of surface-bound and internalized AuNP-aptamer-STAT3d constructs in FaDu and A431 cells. The % of total in the bound and internalized fractions was determined by measuring fluorescence of cell lysates after fractionation (surface and internalized). AuNP constructs were incubated in the cell culture for 4h at 37^oC. C- Internalization of STAT3d, AuNP-STAT3d and AuNP-NUAP-STAT3d in FaDu and A431 cells by [^99mTc] radiolabeling of a one of the strand of STAT3d by using modification with MAG3 chelate.

A - a scheme showing a gold nanoparticle with cell-surface specific nucleolin aptamer (NUAP) that forms a quadruplex dimer and Alexa Fluor 568 labeled STAT3-binding duplex (STAT3d); B - transmission electron microscopy of AuNPs, bar = 100 nm; C- a pseudo-color fluorescent image of a polyacrylamide gel (10% TBE) showing electrophoretic analysis of AuNP-ODN constructs and their components, lane: 1) STAT3d; 2) AuNP-STAT3d; 3) AuNP-CTAP-STAT3d; 4) AuNP-CTAP; 5) CTAP. Control aptamer CTAP was labeled with Cy5.5 (red) and one of the strands of STAT3d was labeled with NIR Dye 800CW (green). An asterisk shows the position of STAT3d-800CW, double asterisk- AuNP conjugates retained at the start; arrow- NUAP-Cy5.5

Expression of nucleolin (cell-surface target protein) and STAT3 in epithelial cancer cell lines. Immunofluorescence using anti-nucleolin antibody showing nucleolin expression in epithelioid carcinoma A431 (A), HNC line FaDu (B); ovarian carcinoma OVCAR3 (C). Secondary FITC-conjugated IgG (green) was used. Nuclei were counterstained with DAPI (blue). D- nucleolin expression in membrane (M) and nuclear (N) fractions of cells. Lanes 1,2 - A431, Lanes 3,4 - FaDu, lanes 5,6- HeLa cells. E- electrophoretic mobility shift assay (EMSA) showing STAT3d alone (lane 1,3) and after adding FaDu cell lysate (lane 2,4). STAT3 d was labeled either using Cy5.5 (lanes 1,2) or dual-labeled with Cy5.5 and 800CW(lanes3, 4); F- Western blotting of STAT3 in FaDu cell extract (lane 1) and HeLa extract (lane 2, positive control).

The effect of RT on viability (A) of A431 (blue bars) and FaDu cells (red bars) and genomic DNA damage (B) pre- (solid bars) and post- (hatched bars) radiation treatment. Both viability and the damage to DNA were determined by using flow cytometry. Control cells (no pre-incubation), or experimental cells incubated with 1) Cetuximab (control as a standard immunotherapy for head and neck cancer), 2) STAT3d or 3) AuNP-NUAP-STAT3d for 20h and then either mock-irradiated or irradiated with a single dose of 4Gy. Staining performed with FITC- labeled rabbit polyclonal anti- γ-H2AX antibodies and analyzed by FACS. * - Indicates statistical significance, p<0.005.