Figure 6.

PSD-A inhibits STAT3 signaling pathway in A549 cells. (a) PSD-A inhibits constitutive STAT3 activation at tyrosine 705. Cells were treated with PSD-A for 24 h, and expression of p-STAT3 and total STAT3 was measured using Western blot. (b) PSD-A inhibits inducible STAT3 activation in A549 cells. A549 cells were pretreated with PSD-A for 4 h and then stimulated with 100 nM TPA and 10 ng/mL IL-6 for 1 h. Extracts were prepared and subjected to Western blot for the expression of p-STAT3 and STAT3. (c) Cells were treated with PSD-A for 24 h, and expressions of phosphatases (STAT3-negative regulators) were determined by Western blot. PSD-A increased the expression of SHP-1 without affecting SHP-2 and PTEN. (d) Cells were treated with 25 nM PSD-A in the presence or absence of Na₃VO₄ (100 μM) for 24 h. Cell lysates were collected and subjected to Western blot for the expression of p-STAT3 and STAT3. Na₃VO₄ pretreatment reversed the suppressive effect of PSD-A on STAT3 indicating that tyrosine phosphatases play an important role in PSD-A-mediated STAT3 inhibition. (e) Cells were treated with PSD-A for 4 h, and expressions of tyrosine kinases (p-SRC/SRC and p-JAK2/JAK2) were measured by Western blot. PSD-A suppressed the phosphorylation of SRC but did not affect p-JAK2 expression. (f) Cells were treated with or without PSD-A in the presence or absence of momelotininb (JAK inhibitor) and SP600125 (JNK inhibitor) for 4 h. Proteins were extracted, and expression of p-STAT3 and STAT3 was detected by Western blot. (g) Cells were treated with PSD-A (50 nM) and S31–201 (100 μM) for 4 h, and expression of p-STAT3 was measured in cell lysates by Western blot. (h) A549 cells were incubated with or without PSD-A for 4 h and then further incubated with IL-6 for 1 h. The nuclear extracts were then collected and assayed for STAT3 DNA-binding activity according to the instructions of the kit. Columns not sharing the same superscript letters within the groups differ significantly (P < 0.05).