Fig. 4

CSMD1 suppressed the activation of the NF-kB and STAT3 pathways, IL-6 and IL-8 secretion. Cells were serum-starved for 2 h followed by a treatment with TNF at a concentration of 25 ng/ml for 2 h. A negative control was established by treating cells with BSA. A & F Representative western blots of fractionated lysates of Ctrl and CSMD1-overexpressing clones of U-118 and U-87 cells immunodetecting pP65-Ser536, total P65, β-actin and H2B. β-actin was used as an internal control for the cytosolic fraction. H2B was used as an internal control for the nuclear fraction. Densitometry analysis of pP65-Ser536/β-actin, total P65/β-actin, pP65-Ser536/H2B and total P65/H2B in (B-E) U-118 and (G-J) U-87 cells after fractionation. K & P Representative western blots of fractionated lysates of Ctrl and CSMD1-overexpressing clones of U-118 and U-87 cells immunodetecting pSTAT3-Y705, total STAT3, β-actin and H2B. Densitometry analysis of p pSTAT3-Y705/β-actin, total STAT3/β-actin, pSTAT3-Y705/H2B and total STAT3/H2B in (L-O) U-118 and (Q-T) U-87 after fractionation. Cells were serum-starved for 2 h followed by a treatment with TNF at a concentration of 25 ng/ml for 2 h. A negative control was established by treating cells with BSA. The amount of secreted (U-W) IL-6 and (X–Z) IL-8 was lower in the CSMD1-overexpressing clone of the glioma cell line after BSA or TNF stimulation. A two-way ANOVA Bonferroni’s multiple comparisons test was used when comparing 3 or more groups with 2 variables (* < 0.05, ** < 0.01, **** < 0.0001)