Fig 2: p-JNK effects on a-SNC secretion do not depend on the transcriptional activity of cJUN. PC12 cells expressing a-SNC/p25a were differentiated with NGF and transduced with control vector or a dominant-negative cJUN-?169-FLAG construct in doses of viral supernatant of 25 or 50 µl before transgene (a-SNC/p25a) induction with doxycycline. a Four days post-infection cells were prepared for immunofluorescence with anti-FLAG antibodies as indicated. Alexa647-conjugated phalloidin (F-actin) was used as counter stain. Bar, 10 µm. b Cell lysates were prepared for western blotting with anti-FLAG mAb for the detection of cJUN-?169-FLAG. Arrow indicates 25 kDa weight marker. The WB is the representative of two independent experiments. c Conditioned medium from transduced cells as above was analyzed for a-SNC by western blotting. All lanes were derived from the same membrane. d Normalized mean and SEM integrated optical density of a-SNC western bands above for four independent experiments

Fig 3: Whole cigarette smoke promoted human IL-1ß and IL-6 secretions through the ERK1/2 MAP kinase and NF?B signaling pathways.Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NF?B inhibitor (IKK-2) for 45 min before exposure or not to cigarette smoke for 15 min. supernatants were collected 24 h later and used to measure the secretion of IL-1ß and IL-6 by ELISA kits. Data are expressed the means+SD, (n = 4).

Fig 4: Whole cigarette smoke promoted human ß-defensin-2 and -3 expression through the ERK1/2 MAP kinase and NF?B signaling pathways.Confluent (80%) gingival epithelial cell cultures were incubated with 10 µM of ERK inhibitor (PD98059), 10 µM of p38 inhibitor (SB202190), 10 µM of JNK inhibitor (SP600125), or 10 µM of NF?B inhibitor (IKK-2) for 45 min before exposure to cigarette smoke for 15 or 30 min. Six hours later, total RNA was extracted, and HBD gene expression was analyzed by qRT-PCR (n = 6). *, p<0.05; **, p<0.01 when comparing values obtained in the presence and absence of inhibitor. The results are expressed as the means+SD, (n = 5).

Fig 5: NED416 activates mitogen-activated protein kinase signaling proteins. (A) Expression of p-ERK, p-JNK and p-p38 protein kinases after NED416 treatment in mouse skin samples. Quantification of the activation of (B) ERK, (C) JNK and (D) and p38 upon NED416 treatment. Data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 vs. 0%. NED416, (E)-3-(2,4-dichlorophenyl)-N-phenylacrylamide; p, phosphorylated.