Down-regulation of ERK1,2 phosphorylation with siRNA or PD98059 does not affect phosphorylation of STAT3 in INA-6 cells. (A) INA-6 cells kept in medium with IL-6 or in coculture with BMSCs, and with or without blockade of the MEK1,2/ERK1,2 module with the pharmacologic inhibitor PD98059 (50 μM) or with siRNAs for ERK1 and ERK2. Note the absence of basal levels of phosphorylated ERK1,2 protein in samples transfected with siRNA expression constructs or incubated with PD98059, and the lack of any effect of these treatments on the phosphorylation of STAT3 at Tyr705. Also note the much stronger phosphorylation of ERK1,2 in cells cocultured with BMSCs, and its absence in samples transfected with pSU-ERK1 + 2 siRNA expression vectors or incubated with PD98059. Equal loading and equal presence of STAT3 were assessed through immunostaining for α-tubulin and STAT3, respectively. (B) INA-6 cells kept in medium with IL-6 or in coculture with BMSCs, and with or without blockade of IL-6R/STAT3 signaling, either with the IL-6R superantagonist Sant7 (50 μg/mL) or an siRNA specific for STAT3. Treatment with Sant7, or expression of the siRNA, led to a strong decrease in the amount of phosphorylated STAT3 in medium with IL-6, but also in the presence of BMSCs. The siRNA caused a near-complete loss of STAT3 protein, whereas Sant7 treatment only affected phosphorylation. These treatments had no effect on the phosphorylation status or expression level of ERK1,2. α-tubulin staining served as loading control.