Figure 2.

LncRNA POU3F3 contributes to MGMT-induced DTIC resistance. (A) Western blot assay for the MGMT expression was performed with lncRNA POU3F3 overexpressing cells, knockdown cells, and control cells. GAPDH was used as the loading control. (B) The transfected cells were pretreated with DTIC (A375 cells: 25 μg/ml; MV3: 10 μg/ml) for 48 h. Cell apoptosis was detected with the flow cytometry analysis. (C) A375/DTIC-silncPOU3F3 cells and control cells were treated with 25 μg/ml DTIC for 48 h and cell apoptosis was analyzed with a flow cytometer. (D) Western blot assay for the MGMT expression was performed with shMGMT transfected A375-lncPOU3F3 cells. (E) The transfected cells were treated with 25 μg/ml DTIC for 48 h and cell apoptosis was analyzed with a flow cytometer. The cell apoptosis data are presented as mean ± SD of at least three independent experiments. (F) The lncRNA POU3F3 overexpressing and controlled A375 cells were subcutaneously implanted for xenografts. DTIC (5 mg/kg) was IP administered every 2 days after the average volume of xenografts was 100 mm³. The volume of the xenografts was recorded every 3 days. (G) The volume of A375-lncPOU3F3 xenografts was compared between A375-vector and A375-lncPOU3F3 ones. (H) The expression levels of MGMT and Bcl-2 were determined by IHC staining with the xenografts as indicated in (G), where *p < 0.01. LncRNA, long non-coding RNAs; DTIC, dacarbazine; MGMT, O6-methylguanine-DNA-methyltransferase; IP, intraperitoneally; IHC, immunohistochemical.