Effects of IL-6R/STAT3 and MAPK pathway blockade on the proliferation of INA-6 cells. (A) INA-6 cells transfected with expression plasmids for siRNAs against ERK1 and ERK2 (pSU-ERK1 + 2) or with control plasmid (pSU), and untransfected cells treated with PD98059, were cultured in medium with IL-6 (left) or in the presence of BMSCs (right) and their proliferation assayed after 3 days. Proliferation (ie, incorporation of [³H]-thymidine) was very similar for cells cultured in medium or on BMSCs with a less than 20% difference in total counts. Within each experimental set the proliferation of unmanipulated cells was arbitrarily set as 100%. Blockade of MEK1,2/ERK1,2 signaling slightly reduced proliferation in both settings. (B) INA-6 cells transfected with an expression plasmid for an siRNA against STAT3 (pSU-STAT3) or with control plasmid (pSU), and untransfected cells treated with Sant7, were cultured in medium with IL-6 (left) or in the presence of BMSCs (right). In keeping with extensive cell death, Sant7 and the siRNA abrogated all proliferation in medium, but proliferation with either treatment was only moderately reduced in the presence of BMSCs. (C) Confirmation of the specificity of STAT3 siRNA-mediated cell death. Expression of STAT3 protein that is derived from a sequence no longer recognizable by the siRNA (plasmid pS3R) largely offset the detrimental effects of STAT3 siRNA expression on the proliferation of INA-6 cells. All data shown are derived from at least 3 independent experiments. Statistical analysis (A,B) revealed very significant (^**), significant (^*), or no (n.s. = not significant) differences as indicated. Error bars denote the range of values derived from at least 3 independent experiments.