Fig. 3.
The role of P2X7 upstream of SFKs in the signaling pathway activated by LL-37 in primary keratinocytes. a, b The cells were pretreated with P2X7 inhibitor KN-62 (5 µM) or an equivalent concentration of methanol vehicle control for 1 h and stimulated with LL-37 (5 µg/ml). a Western blots of cell lysates at 15 min of stimulation analyzed for phospho-Src Y416 (Src pY416), total Src and GAPDH loading control. b Quantification of the phospho-Src Y416 blots using ImageJ 1.43u software, band intensities normalized against GAPDH loading control and shown as fold change relative to the intensity of the untreated sample. All whiskers show means ± SEM from 4 independent experiments; * p < 0.05 using paired t test; analysis by ANOVA with Dunnett's post hoc test showed significant induction of phospho-Src Y416 levels in the control but not the inhibitor-treated samples. c, d The cells were pretreated with the Gi-protein coupled receptor inhibitor PTX (100 ng/ml) or an equivalent concentration of DMSO vehicle control for 1 h and stimulated with LL-37 (5 µg/ml). c Western blots of cell lysates at 15 min of stimulation analyzed for phospho-Src Y416 and GAPDH loading control. d Quantification of the phospho-Src Y416 blots, band intensities normalized against GAPDH loading control and shown as fold change relative to the intensity of the untreated sample, data from 5 independent experiments. e Cell viability at 24 h of stimulation with LL-37 (5 µg/ml) alone or in combination with KN-62 (5 µM), PTX (100 ng/ml), or vehicle controls, measured using the WST1 assay and expressed relative to the viability of untreated cells (defined as 100%) and cells treated with 1% Triton-X (defined as 0%).