Fig. 4: DNMT3B suppressed by gefitinib decreased PFTK1 in NSCLC.

a MSP assays were performed to evaluate the methylation status of the PFTK1 promoter in gefitinib-resistant and parental cells. “Methylation” was abbreviated as “M”, “Unmethylation” was abbreviated as “U”. b Six pairs of NSCLC tissues [before and after gefitinib treatment, we abbreviated as sensitive (S) and resistance (R)] were collected, and the methylation status of the PFTK1 promoter was determined by MSP assays. c 5-Azacytidine (5-Aza), an inhibitor of DNMTs, was used to treat HCC827 and PC9 cells for 24 h. The expression of PFTK1 was measured by qRT-PCR assays (n = 3). **p < 0.01. d The relative expression levels of common DNMTs in gefitinib-resistant and parental cells were evaluated by qRT-PCR assays (n = 3). **p < 0.01. e The protein levels of DNMT3B in gefitinib-resistant and parental cells were assessed via WB assay (n = 3). **p < 0.01. f, g DNMT3B overexpression plasmid and control plasmid were transfected into HCC827/GR and PC9/GR cells (f). ShRNAs targeting DNMT3B were transfected into HCC827 and PC9 cells (g). WB and qRT-PCR assays were carried out to estimate the expression level of PFTK1 (n = 3). **p < 0.01. h The occupancy of DNMT3B in the promoter of PFTK1 among gefitinib-resistant and parental cells was valued via ChIP assay (n = 3). **p < 0.01. i The DNMT3B expression in 67 cases of NSCLC tissues with gefitinib treatment was determined by IHC assay. The relationship between DNMT3B and PFTK1 was analyzed. Scale bars: 40 μm (**p < 0.01).