Figure 1.

Up-regulated expression of ABCB1 in the cells and tissues of lung adenocarcinoma. (A) Bioinformatic analysis of ABCB1 levels in the tissues of normal lung (n = 390), lung adenocarcinoma (n = 261) and seven lung adenocarcinoma subtypes. *P < 0.05 vs. normal lung tissues. (B) ABCB1 protein levels in HBEC, H446, H1299, H1975 cell lines analyzed using western blot. n = 3, ***P < 0.001. (C) ABCB1 mRNA expression levels in lung tumor samples and non-tumor samples analyzed using semi-quantitative RT-PCR. n = 20, *P < 0.05. (D, E) Qualitative and quantitative analyses of ABCB1 expression in the lung tumor and non-tumor tissues with immunohistochemical (IHC) staining. n = 50, **P < 0.01. ABCB1: ATP binding cassette subfamily B member 1."

Figure 2.

Up-regulation of ABCB1 expression in the tracheal epithelial cells and lung tissues of GPRC5A knockout mice. (A) Western blot analysis of ABCB1 expression in MTEC-WT cells and MTEC-KO cells taken from 2-month-old GPRC5A+/+ mice and GPRC5A-/- mice respectively. n = 5, ***P < 0.001. (B) Twelve months after injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), western blot was used to analyze the ABCB1 expression in MTEC-WT cells and MTEC-KO cells taken from 14-month-old GPRC5A+/+ and GPRC5A-/- mice. n = 5, **P < 0.01. (C, D) Qualitative and quantitative analyses of IHC staining results of the expression of ABCB1 in the lung tissues of 2-month and 14-month-old GPRC5A+/+ and GPRC5A-/- mice and 14-month-old NNK treated GPRC5A+/+ and GPRC5A-/- mice. n = 5, *P < 0.05, **P < 0.01. KO: knock out; WT: wild type; NNK: 4-(methylntrosamino)-1-(3-pyridyl)-1-butanone; GPRC5A: G protein coupled receptor family C group 5 type A."

Figure 3.

Cell viability of MTEC-KO and MTEC-WT cells treated with tariquidar (A) or doxorubicin (B) analyzed with cell counting kit-8. n=3, **P < 0.01."

Figure 4.

The improving effect of ABCB1 knockdown on the progression of lung tumors in BALB/c nude mice. (A) Western blot analysis of ABCB1 expression in four ABCB1 knockdown SJT-1601 cells and SJT-1601-wild type (WT) cells. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001. (B, C) Tumor volume and tumor weight in BALB/c nude mice after transplantation of SJT-1601-k1 and SJT-1601-k4 cells for 28 days compared to mice transplanted with SJT-1601-WT cells. n = 5; **P < 0.01."

Figure 5.

Expression of ABCB1 reduced by GPRC5A in tracheal epithelial cells of GPRC5A knockdown mouse (MTEC-KO). (A) RT-PCR analysis of ABCB1 and GPRC5A mRNA levels in MTEC-WT (wild type tracheal epithelial cells), MTEC-KO, and MTEC-KO-m5a (MTEC-KO transfected with GPRC5A) cell lines. n = 3, ***P < 0.001. (B) Western blot analysis of ABCB1 and GPRC5A protein levels in MTEC-WT, MTEC-KO, and MTEC-KO-m5a cell lines. n = 3, ***P < 0.001. (C) Immunofluorescent analysis was used to observe the co-localization of ABCB1 (green) with GPRC5A (red) in HCC827 cell line. (D) HCC827 cells were co-transfected with Flag-ABCB1 plasmid plus empty or Myc-GPRC5A plasmid, cell lysates were processed by magnetic bead method, and finally subjected to western blot assay."