Table 1.
The real-time PCR primers sets.
Fig 1.
The mRNA expression of PD-L1 and PD-L2 in breast cancer cell lines.
The relative mRNA expressions of PD-L1 (A) and PD-L2 (B) on breast cancer cell lines were measured by qPCR (n = 4 each). The values were relative to the mRNA expressions of SK-BR-3. Each mean ± SEM is shown. N.D: Not detected.
Fig 2.
The effect of nicotine treatment for PD-L1 expressions on breast cancer cell lines.
(A-C) Breast cancer cell lines, HCC1937 cells (A), MDA-MB-231 cells (B), and SK-BR-3 cells (C) were stimulated with nicotine (0–100 nM) and the relative mRNA expressions of PD-L1 to those without the stimulations (nicotine 0 nM: Relative expression = 1) were measured by qPCR (n = 4 each). (D, E) SK-BR-3 cells were cultured in the absence and presence of nicotine (1, 10 and 100 mM) and were stained with anti-PD-L1 antibody. DAPI was used for detecting nuclei. (D) One of the representative figures is shown. (E) The relative expression of PD-L2 was determined by using ImageJ (n = 5 each). Each mean ± SEM is shown. *p < 0.05 and ***p < 0.001.
Fig 3.
The effect of nicotine treatment for PD-L2 expressions on breast cancer cell lines.
(A-C) Breast cancer cell lines, HCC1937 cells (A), MDA-MB-231 cells (B), and SK-BR-3 cells (C) were stimulated with nicotine (0–100 nM) and the relative mRNA expressions of PD-L2 to those without the stimulations (nicotine 0 nM: Relative expression = 1) were measured by qPCR (n = 4 each). (D, E) SK-BR-3 cells were cultured in the absence and presence of nicotine (1, 10 and 100 mM) and were stained with anti-PD-L2 antibody. DAPI was used for detecting nuclei. (D) One of the representative figures is shown (n = 5 each). (E) The relative expression of PD-L2 was determined by using ImageJ. Each mean ± SEM is shown. *p < 0.05 and **p < 0.01.
Fig 4.
Nicotine decreased Wnt5a expressions in SK-BR-3 cells.
(A) Cell viability of SK-BR-3 cells with nicotine treatment (0–100 nM) was measured by MTT assay (n = 4 each). (B, C) KLF4 (B) and Wnt5a (C) mRNA expressions in SK-BR-3 cells at 24 h after nicotine treatment were measured by qPCR (n = 4 each). Each mean ± SEM is shown. ***p < 0.001.
Fig 5.
Nicotine decreased Akt phosphorylation in SK-BR-3 cells.
(A) Akt expressions and the phosphorylation in nicotine-treated SK-BR-3 cells were assayed by immunostaining. SK-BR-3 cells were stimulated with 100 nM nicotine for indicated time and the cells were stained with indicated antibodies (red). DAPI (blue) was used for detecting nuclei. Some of representative figures of total-Akt (upper), phosphorylated Akt (lower) are shown. The relative phosphorylation intensity of Akt was calculated using ImageJ (n = 5–7 each). (B-D) Akt expressions and the phosphorylation in nicotine-treated MCF8 (B), HCC1937 (C) and MDA-MB-231 (D) cells were assayed by immunostaining. SK-BR-3 cells were stimulated with 100 nM nicotine for indicated time and the cells were stained with indicated antibodies (red). DAPI (blue) was used for detecting nuclei. Some of representative figures of total-Akt (left), phosphorylated Akt (right) are shown (n = 3–4 each). (E, F) The PD-L1 (E) and PD-L2 (F) expressions in MK-2206, an Akt inhibitor, -treated SK-BR-3 cells are shown. SK-BR-3 cells were stimulated with MK-2206 (0–50 nM) for 24 h and the cells were stained with indicated antibodies (red). DAPI (blue) was used for detecting nuclei. Some of representative figures of PD-L1 or PD-L2 (left) and the relative expression levels (right) are shown. The relative expression levels of PD-L1 or PD-L2 are determined by using ImageJ (n = 5–7 each).
Fig 6.
The expression of nAChR subunits in breast cancer cells.
The relative mRNA expressions of nAChR subunits in breast cancer cell lines was measured by qPCR. The values were relative to the mRNA expressions of MCF7 cells. N.D.: Not detected.