Fig 2: A schematic molecular model of nicotine-induced α9-nAChR activity in melanoma cells. α9-nAChR mediates nicotine-induced PD-L1 expression and regulates melanoma cell proliferation and migration. Nicotine-induced α9-nAChR activity upregulates Snail-1 and Twist-1 expression and activates the phosphorylation of AKT, ERK, and STAT3.

Fig 3: α9-nAChR mediates nicotine-induced PD-L1 expression and regulates melanoma cell proliferation. (A) Nicotine increased STAT3, AKT, and ERK phosphorylation in a time-dependent manner in the A375, A2058, and MDA-MB-435 cells, as assessed by western blotting. (B) Nicotine induced the phosphorylation of STAT3, AKT, and ERK and upregulated the protein levels of α9-nAChR, PD-L1, Snail-1 and Twist-1 in a dose-dependent manner. The results were determined by western blotting. (C–J) A2058 cells were pretreated with the inhibitor LY294002 (10 μm or 25 μm), PD98059 (10 μm or 25 μm), Stattic (2.5 μm or 5 μm) or NSC74859 (10 μm or 25 μm) for 3-24 h and subsequently treated with or without nicotine (1 μm) for additional 24-48 h. Western blot analysis was used to detect protein levels. Cell proliferation was assessed by an MTT assay. (K,L) Wild-type, scrambled siRNA, and α9-nAChR-si-expressing A2058 cells were treated with or without 1 μm nicotine. Western blot analysis was used to assess protein levels. Cell growth was assessed by an MTT assay. (M,N) After A2058 cells were exposed to 0.1 μm or 1 μm nicotine for 24 h, a ChIP assay was performed using an anti-STAT3 antibody. The protein-chromatin immunoprecipitates were subjected to RT-PCR and qPCR. In RT-PCR, a rabbit IgG antibody was used as a negative control, and total genomic DNA was used as a positive control. In qPCR, relative enrichments of PD-L1 promoter region were calculated after normalization to GAPDH. Each of the immunoprecipitations was replicated three times, and each sample was quantified at least in triplicate. The data are presented as the mean ± SD of three independent experiments. ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; Student’s t-test.

Fig 4: IAV Induces miR-1290 Expression by Activation of the ERK Pathway(A) Comparison of miR-1290 expression in A549 cells infected with either WSN (wild type) or UV-inactivated WSN (UV) at an MOI of 2 for 2 h. (B) Comparison of miR-1290 expression in mock-infected or WSN-infected A549 cells treated with either ddH2O or NH4Cl. (A and B) Experiments were performed in triplicate and repeated at least three times. Data are presented as mean values ± SD from one representative experiment. (C) Validation of ERK phosphorylation in WSN-infected A549 cells. A549 cells were infected at an MOI of 5. Experiments were performed at least three times. Data are presented from one representative experiment. (D) Comparison of miR-1290 expression in DMSO- or TPA-treated A549 cells. qPCR was performed in triplicate and repeated at least three times. Data are presented as mean values ± SD from one representative experiment. Western blotting was performed at least three times. Data are presented from one representative experiment. (E) Validation of ERK phosphorylation in A549 cells infected with different IAV strains. A549 cells were infected at an MOI of 5 for 0.5 h. Western blotting was performed at least three times. Data are presented from one representative experiment. (F) Comparison of miR-1290 expression in A549 cells infected with different IAV strains at an MOI of 5 for 2 h. Experiments were performed in triplicate and repeated at least three times. Data are presented as mean values ± SD from one representative experiment. (C–E) After western blot analysis, ERK activation was analyzed with a monoclonal antibody specific to phosphorylated ERK (p-ERK). Loading levels were controlled with a polyclonal antibody against ERK2. U6 was used as an internal control, and each sample was normalized with a mock control. Error bars represent the SD derived from three independent experiments. Statistical significance was determined by conducting an unpaired t test (*p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001; ns, nonsignificant). H.P.I., hours post infection.

Fig 5: α9-nAChR overexpression induces PD-L1 upregulation and promotes melanoma cell proliferation and migration. (A) Overexpression of α9-nAChR activated the phosphorylation of AKT, ERK, and STAT3 and induced PD-L1, Snail-1, and Twist-1 protein levels in A2058 and MDA-MB-435 cells, which were evaluated by western blotting. (B,C) α9-nAChR overexpression significantly increased the proliferation of A2058 and MDA-MB435 cells compared to control cells, as determined by a cell count assay. (D) Soft-agar growth assays were performed with A2058 and MDA-MB-435 cells transfected with pcDNA3.1 or pcDNA3.1-α9-nAChR overexpression vectors. (E) Colony numbers were quantified and presented in the histogram. (F) Cell morphology of A2058 and MDA-MD-435 cells transfected with pcDNA3.1 or pcDNA3.1-α9-nAChR overexpression vector. (G,H) Representative micrographs and statistical analysis demonstrated that α9-nAChR overexpression increased the migration areas of A2058 and MDA-MB 435 cells at 24 h after scratching. (I,K) Percentages of cell proliferation for the A2058 cells, pcDNA3.1-A2058 cells, and α9-nAChR-overexpressing A2058 cells were treated with or without the PI3K inhibitor LY294002 (10 µm) or MEK inhibitor PD98059 (10 µm) for 48 h determined by an MTT assay. (J,L) Western blotting to detect the protein levels of P-AKT and P-ERK in pcDNA3.1-A2058 cells and α9-nAChR-overexpressing A2058 cells with or without LY294002 or PD98059 treatment. The data are presented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.