Fig 2: Effect of conditioned context on the coexpression of c-Fos, Arc, and FG in BLA projection neurons to the PrL.(A) First line: c-Fos–positive neurons in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups (green-colored). Second line: Arc-positive neurons in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups (red-colored). Third line: FG-labeling neurons in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups (blue-colored). Fourth line: Colabeling neurons of c-Fos and FG (cyan-colored); colabeling neurons of c-Fos, Arc, and FG (white-colored); and colabeling neurons of c-Fos and Arc (yellow-colored) in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups. Scale bars, 100 μm. BLA regions enclosed by white boxes were shown in a higher magnification in upper right square images. Scale bars, 20 μm. (B) Left: Average percentage of the expression of c-Fos colabeled with FG, but not with Arc, relative to FG in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups (n = 5 mice in saline + saline, saline + naloxone, and morphine + saline groups; n = 7 mice in morphine + naloxone group; one-way ANOVA, *P < 0.05). Middle: Average percentage of the expression of c-Fos, Arc, and FG relative to FG in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups (n = 5 mice in saline + saline, saline + naloxone, and morphine + saline groups; n = 7 mice in morphine + naloxone group; one-way ANOVA, P > 0.05). Right: c-Fos– and Arc-positive neurons/mm2 in the BLA in saline + saline, saline + naloxone, morphine + saline, and morphine + naloxone groups (n = 5 mice in saline + saline, saline + naloxone, and morphine + saline groups; n = 7 mice in morphine + naloxone group; one-way ANOVA, *P < 0.05). Data are means ± SEM.

Fig 3: c-Jun Ser73 phosphorylation is essential for ERK-dependent demethylation and activation of Zta promoter.(a) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA for 1, 3, 6, 12, and 24 h, respectively. Whole-cell lysates were isolated and the levels of phosphorylated ERK (p-ERK), phosphorylated c-Jun (p-c-Jun), phosphorylated c-Fos (p-c-Fos), ERK, c-Jun, c-Fos, Zta, and GAPDH proteins were determined by Western blotting using corresponding antibodies. (b) B95-8 cells were pretreated with U0126 (20 μM) for 2 h and then treated with TPA for 15 and 90 min. Whole-cell lysates were isolated and the levels of phosphorylated c-Jun (p-c-Jun) and GAPDH proteins were determined by Western blotting. (c) B95-8 cells were treated with TPA for 0.5, 1, 3, 6 and 9 h. Total RNAs were isolated from the cells and the level of Zta mRNA was analyzed by qPCR. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 4: The Roles of LNAPPCC in Promoting Colon Cancer Cell Proliferation and Migration Are Dependent on the LNAPPCC/PCDH7/ERK/c-FOS Feedback Loop(A) Cell proliferation of LNAPPCC stably overexpressed HCT116 cells treated with 1 μM GDC-0994 was examined by CCK-8 assays. (B) Cell proliferation of LNAPPCC stably overexpressed HCT116 cells treated with 1 μM GDC-0994 was examined by EdU incorporation assay. Scale bars represent 100 μm. (C) Cell migration of LNAPPCC stably overexpressed HCT116 cells treated with 1 μM GDC-0994 was examined by Transwell migration assay. Scale bars represent 100 μm. (D) PCDH7 expression levels in LNAPPCC stably overexpressed and concurrently PCDH7 stably silenced HCT116 cells. (E) Cell proliferation of LNAPPCC stably overexpressed and concurrently PCDH7 stably silenced HCT116 cells was examined by CCK-8 assays. (F) Cell proliferation of LNAPPCC stably overexpressed and concurrently PCDH7 stably silenced HCT116 cells was examined by EdU incorporation assay. Scale bars represent 100 μm. (G) Cell migration of LNAPPCC stably overexpressed and concurrently PCDH7 stably silenced HCT116 cells was examined by Transwell migration assay. Scale bars represent 100 μm. Data are presented as mean ± SD based on at least three independent biological repeats. ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant, by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Fig 5: Inhibitory effect of the phosphorylation of the JNK, p38 MAPK, and ERK1/2, and the transcription factors c-Jun and c-Fos, in UV-B-exposed and NK-CdM-treated NHDFs. After being serum-starved for 24 h, NHDFs were irradiated with UV-B (30 mJ/cm2), and further incubated with NK-CdM at the indicated concentrations for 48 h. Total cell protein extracts were prepared and separated by electrophoresis on SDS-PAGE gels followed by a western blot analysis with primary antibodies capable of detecting (A) phospho-JNK, -p38 MAPK, and -ERK1/2 and (B) phospho-c-Jun, -c-Fos. β-actin was used as the loading control. (C) Equal protein loading was verified by analyzing β-actin levels. (D) The antioxidant activity of NK-CdM, at the indicated concentration, was measured using an Antioxidant Assay kit. (E) NHDFs were treated with 10 μM of the ERK1/2 inhibitor PD, 10 μM of the JNK inhibitor SP, 10 μM of the p38 MAPK inhibitor SB, or 10 μM of the PI3K inhibitor LY and then irradiated. The levels of procollagen I and type I collagen at 48 h after irradiation were measured by western blot analysis. (F) The bar graphs (mean ± standard error of the mean; n=3) represent a quantitative densitometric analysis of the bands. #P<0.05 and ###P<0.001 vs. the Normal (non-irradiated control). **P<0.01 and ***P<0.001 vs. the UV-B-irradiated control. $P<0.05, $$P<0.01 and $$$P<0.001 vs. the UV-B irradiated and 1.25% NK-CdM treated group. MMP, matrix metalloproteinase; ERK, extracellular signal regulated kinase; NK-CdM, natural killer cell conditioned medium; NHDF, neonatal human dermal fibroblasts; UV, ultraviolet; MAPK, mitogen associated protein kinase; PI3K, phosphatidylinositol 3 kinase; SP, SP600125; LY, LY294002; SB, SB203580; PD, PD98059; phosphor, phosphorylated; JNK, c-Jun N terminal kinase.