STAT3 is constitutively active during cervical carcinogenesis. (A) Cervical cancer cell lines C33a, SiHa and CaSki express constitutively active STAT3 DNA-binding activity. Ten μg of nuclear proteins of respective cell line were incubated with 32P-radiolabeled STAT3 probe and resolved on 6.0% non-denatunating PAGE. (B) Gel supershift assay showing specificity of STAT3 binding. SiHa cell nuclear proteins (10 μg) were incubated for 30 min with pSTAT3(Y705) or pSTAT3(S727) antibodies (2 μg each) or pre-immune serum (PIS) and assayed for STAT3 binding activity by EMSA. STAT3-specific complex and super-shifted bands after antibody addition is indicated. (C) Constitutive STAT3 activation in cervical precancer and cancer lesions. Nuclear proteins (10 μg) prepared from normal (N1 & N2*), precancer (LSIL and HSIL) and cancer lesions (Ca1 & 2) were tested for presence of STAT3 DNA binding activity by EMSA. The extent of STAT3 activity increased as a function of severity of the disease. Cancerous lesions showed high STAT3 binding activity. N2* showed inflammatory cytology. (D) Nuclear proteins were simultaneously checked for equal loading using EMSA for constitutively active transcription factor Oct-1 that showed uniform binding in different tissues types. (E) Mean fold change in STAT3 DNA binding activity in cervical precancer and cancer lesions with respect to normal controls. Error bars indicate standard deviation. ap value < 0.001 versus normal control tissues; bp value < 0.001 versus precancer tumor tissues as determined by t-test.