Fig. 7

Pretreatment with LPS followed by CA140 treatment decreased phosphorylation of STAT3 in the nucleus and cytosol. a BV2 microglial cells were pretreated with LPS (1 μg/mL) or PBS for 45 min, followed by treatment with vehicle (1% DMSO) or CA140 (10 μM) for 5.5 h and subcellular fractionation (nuclear and cytosolic fractions). Western blotting was performed on the cytosolic fraction using antibodies against p-STAT3 (Ser727) and β-actin. b Quantification of data from a (con, n = 12; LPS, n = 12; LPS + CA140, n = 12). c, d Western blotting was performed on the nuclear fraction using anti-p-STAT3 (Ser727) and anti-PCNA antibodies (con, n = 12; LPS, n = 12; LPS + CA140, n = 12). e, f BV2 microglial cells were pretreated with LPS (1 μg/ml) or PBS for 30 min, followed by treatment with vehicle (1% DMSO) or CA140 (10 μM) for 5.5 h and immunostaining with anti-p-STAT3 (Ser727) and anti-CD11b antibodies (con, n = 202; LPS, n = 169; LPS + CA140, n = 397). g–i BV2 microglial cells were pretreated with LPS (1 μg/mL) or PBS for 30 min, followed by treatment with a STAT3 inhibitor (S3I-301, 10 μM) or vehicle (1% DMSO) for 30 min and finally CA140 (10 μM) or vehicle (1% DMSO) for 5 h. Total RNA was then isolated, and IL-1β or COX-2 mRNA levels were measured by RT-PCR (COX-2 and IL-1β: con, n = 17; LPS, n = 17; LPS + CA140, n = 17). *p < 0.05, **p < 0.01, ***p < 0.0001