Figure 5. Effect of STAT3 overexpression in FT33 cells in vitro:

(A) ICC of normal FT 33 cells displayed pSTAT3 Tyr705 expressions in the cytoplasm of the cells as evident by the green fluorescence. c-MYC was also present in the cytoplasm of a few cells. All the cells of interest are marked by yellow arrows. (B) Confirmation of STAT3 overexpression in FT33 cells using a Western Blot where FT is control and FT OE is STAT3 overexpression. The bottom panel is his-tag expression which is tagged to STAT3 gene in the plasmid. In order to create STAT3 overexpression cells, FT33 cells were transfected with a vector harboring STAT3 gene or with the empty vector backbone for control. (C) Reverse Transcription qualitative PCR confirmed the relative expression of STAT3 and associated genes using RNA extracted from normal fallopian tube cells (FT) and fallopian tube cells overexpressing STAT3 (FTOE). The RNA was converted to cDNA and amplified with gene specific primers; relative expression of STAT3, c-Myc and cyclin D1 goes up while PIAS3 and StIP1 go down (values normalized to GAPDH; ***p<0.0005, ** p<0.005, *p<0.05). Immunocytochemistry (ICC) of control FT 33 and FT 33 STAT3 OE cells with pSTAT3 Tyr705 (left) and c-myc (right). (D) Quantification of scratch assay showing percent migration of control FT33 cells in comparison to FT33 cells overexpressing STAT3. (E & F) On an intracellular level, FT cells overexpressing STAT3 displayed a more nuclear presentation for both pSTAT3 Tyr705 and c-Myc (green fluorescence for the genes against the blue nuclear background).