Berberine could suppress the STAT3 activation in NPC cells induced by IL-6 secreted from fibroblasts. (a) IL-6 was secreted by tumor-associated fibroblasts. 1 x 106 fibroblasts were seeded in each well of a 6-well plate. Fibroblast supernatant was collected after 1, 2, 3 and 4 days of incubation in culture hood. The concentration of IL-6 was assessed by ELISA. (b) Fibroblast supernatant (FS) could activate the STAT3 signaling in C666-1 cells. FS collected after 4 days of incubation was used to treat C666-1 for 1, 2, 4 and 6 hr. Increasing levels of p-STAT3 could be detected in a time-dependent manner. (c) Berberine could inhibit the activation of STAT3 induced by FS. 50 μM of berberine could effectively suppress the FS-induced STAT3 activation at 4 hr time points. Three independent experiments were carried out, relative ratio to β-actin was calculated, and data are represented as mean ± S.D. from three experiments. *, p < 0.05. (d) The activation of STAT3 in FS-treated NPC cells was dependent on the IL-6\IL-6R signaling pathway. C666-1 cells were pretreated with anti-IL-6 or anti-IL-6R antibodies for 30 mins before the addition of FS. Protein was collected after 4 hr treatment of FS. Anti-IL-6 or anti-IL-6R antibodies could significantly suppressed the FS-induced activation of STAT-3. Three independent experiments were carried out, relative ratio to β-actin was calculated, and data are represented as mean ± S.D. from three experiments. *, p < 0.05. (e) IL-6 could enhance the growth rate of C666-1 cells. The cells were treated with IL-6 (20 ng/ml) or pretreated with anti-IL-6 antibody for 30 mins before the addition of IL-6. After 24 hr, the growth rate was determined by MTT assay. Mean ± SD were calculated from triplicate of two independent experiments. *, p < 0.05.