The involvement of NF-κB activation in IL-1β-mediated-MMP-3 expression.

(a) The effect of IL-1β (100 pM, 24 h) on MMP-3 mRNA expression in melanoma cells with or without the NF-κB inhibitor, TPCA-1 (10 μM, 1 h). TBP was used as a reference, and the relative expression levels of MMP-3 mRNA in IL-1β-stimulated melanoma cells were compared with that of 0 h. The results are shown as mean ± standard error (SE) of biological triplicates. *PP<0.05.</p

S1 Raw images -

(PDF)</p

The effect of IL-1β on intracellular pH (a) and the mRNA expression of NHE1 (b).

The cells were treated with (closed) or without (open) 100 pM IL-1β. IL-1β failed to induce the changes in intracellular pH (a, n = 20 cells, randomly selected ×20 fields from triplicate samples) and mRNA expression of NHE1 (b). Data are shown as the mean ± standard error of three independent experiments. (PDF)</p

Effect of the NF-κB inhibitor on the IL-1β-induced phosphorylation of p65/RelA and p105.

The cells were pretreated with the NF-κB inhibitor, TPCA-1 (10 μM, 1 h), followed by the stimulation of IL-1β (100 pM) for 15 min. Phosphorylated p65/RelA (p-p65) and p105 (p-p105), and total p65/RelA (t-p65) and p105 (t-p105) were detected by Western blotting. Representative images of the inhibitory effect of TPCA-1 on IL-1β-induced phosphorylation of p65/RelA (a) and p105 (c) are shown. The relative levels of [p-p65]/[t-p65] (b) and [p-p-105]/[t-105] (d) relative to levels without the inhibitor and IL-1β are illustrated. The results are represented as mean ± SE of biological triplicates. *P<0.05.</p

Expression of MMP-3 and cellular migration in IL-1β-treated canine melanoma cells.

MMP-3 activity in the culture medium (a) and MMP-3 mRNA expression (c) of canine melanoma cells in the presence (closed circle) or absence (open circle) of canine recombinant IL-1β (100 pM). MMP-3 activity in the culture medium (b) and MMP-3 mRNA expression (d) in melanoma cells incubated with IL-1β (0 to 100 pM) for 24 h. TBP was used as a reference. Relative mRNA expression of MMP-3 in IL-1β-treated melanoma cells was compared with the expression at 0 h. (e, f) The cells were treated with (closed) or without (open) 100 pM IL-1β for 24 h in the presence (square) or absence (circle) of UK356618 (50 nM), an MMP-3 inhibitor, for 2 h. The representative data of cell migration (e) and wound area (f) values are shown. The data are represented as the mean ± standard error (SE) of three biological replicates. *P<0.05.</p

Attenuation of IL-1β-induced MMP-3 mRNA expression in other sets of melanoma cells transfected with p65/RelA siRNA, but not with p105 siRNA.

Melanoma cells (a, CMe1; b, eCMMe2; c, LMe) transfected with p65/RelA, p105, or scrambled siRNAs following the stimulation of IL-1β (100 pM) for 24 h. Relative expression of MMP-3 mRNA was evaluated. TBP was used as a reference. The attenuation of MMP-3 expression was observed in p65/RelA-depleted cells but not in the cells transfected with p105 siRNA or scrambled siRNA. The results are represented as mean ± standard error (SE) of biological triplicates. *P<0.05.</p

Sequences for siRNA transfection.

Sequences for siRNA transfection.</p

Primer sequences for RT-qPCR.

Primer sequences for RT-qPCR.</p

Attenuating IL-1β-induced MMP-3 mRNA expression in melanoma cells transfected with p65/RelA siRNA, but not with p105 siRNA.

(a-c) The effect of the depletion of t-p65/RelA, t-105, and β-actin in melanoma cells transfected with p65/RelA, p105, or scrambled siRNAs. Representative images of Western blotting (a) and relative density of the expression of t-p65/RelA (b) or t-p105 (c) in each siRNA-transfected cell compared with those in the cells transfected with scramble siRNA. Depletion of p65/RelA or p105 was observed in melanoma cells transfected with p65/RelA or p105 siRNA, respectively, but not in the cells transfected with scrambled siRNA. β-actin was used as a reference. (d) The effect of siRNA transfection for p65/RelA or p105 on the MMP-3 expression in IL-1β-treated cells. Melanoma cells transfected with p65/RelA, p105, or scrambled siRNAs following the stimulation of IL-1β (100 pM) for 24 h. Relative expression of MMP-3 mRNA was evaluated. TBP was used as a reference. The attenuation of MMP-3 expression was observed in p65/RelA-depleted cells but not in the cells transfected with p105 siRNA or scrambled siRNA. The results are represented as mean ± standard error (SE) of biological triplicates. *P<0.05.</p

The effect of IL-1β on cellular invasion (a) and adhesion (b).

a, In melanoma cells, cellular invasion was undetectable. b, Cellular adhesion of IL-1β, UK356618, and UK356618+ IL-1β-treated cells showed no significant difference compared to control. Data are shown as the mean ± standard error of three independent experiments. (PDF)</p