Fig 2: Western blot analysis of RFPL3, hTERT, ERK and p-ERK protein expression in A549 and H1299 cells, treated with or without MEK-specific inhibitor (PD98059). (A) A549 and H1299 cells were treated with PD98059 (50 µM) for 2 h, and then with 20 ng/ml EGF for 48 h; expression levels of RFPL3, hTERT, ERK and p-ERK proteins were then detected by western blot analysis. PD98059 decreased expression of p-ERK without affecting total ERK protein, and also markedly decreased EGF-induced upregulation of RFPL3 and hTERT in H1299 cells, but did not significantly decrease hTERT expression. (B and C) Histograms indicate relative quantitative expression of these proteins. Data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05 EGF, epidermal growth factor; RFPL3, Ret finger protein like 3; hTERT, human telomerase reverse transcriptase.

Fig 3: Western blot analysis of RFPL3 and hTERT expression in A549 and H1299 cells stimulated with different concentrations of EGF for 24 h or 48 h. (A) A549 and (B) H1299 cells were respectively treated with different concentrations of EGF (0, 2.5, 5, 10, 20 or 50 ng/ml) for 24 h or 48 h. (C-F) Expression of RFPL3 and hTERT proteins were detected by western blot analysis. Results show that EGF stimulation significantly increased RFPL3 protein expression in the A549 and H1299 cells, with corresponding changes in hTERT expression. At 48 h, RFPL3 and hTERT proteins were expressed incrementally in a dose-dependent manner; 20 ng/ml was the optimal concentration. EGF, epidermal growth factor; RFPL3, Ret finger protein like 3; hTERT, human telomerase reverse transcriptase.

Fig 4: Expression of hTERT and related proteins in the MEK signaling pathway in A549 and H1299 cells transfected with an RFPL3-overexpression plasmid. (A) Western blot analysis was used to detect expression of RFPL3, hTERT, Ras, Raf, ERK and p-ERK. Compared with the blank control and negative control groups, RFPL3 in transfected cells was overexpressed, and expression of hTERT, Ras, Raf, ERK and p-ERK also increased. (B and C) Histograms indicate relative quantitative expression of these proteins. Data are expressed as the mean ± standard deviation of three independent experiments. *P<0.05, compared with the negative control group. RFPL3, Ret finger protein like 3; hTERT, human telomerase reverse transcriptase.

Fig 5: mRNA levels of RFPL3 and hTERT in A549 and H1299 cells as detected by RT-qPCR. (A-D) A549 and H1299 cells were treated with different concentrations of EGF (0, 2.5, 5, 10, 20 or 50 ng/ml) for 48 h. Expression of RFPL3 and hTERT mRNA was determined by RT-qPCR. Results showed that EGF markedly increased RFPL3 and hTERT mRNA expression, consistent with the increased protein expression. EGF, epidermal growth factor; RFPL3, Ret finger protein like 3; hTERT, human telomerase reverse transcriptase.