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. 2016 Feb 10;6:20508. doi: 10.1038/srep20508

Figure 5. shRNA-OCT1 downregulation in TOV2223G cells diminishes DNR uptake and confers resistance to the drug.

Figure 5

(A) Western blot analysis. Total cell extracts were prepared from stable clones of TOV2223G cells carrying either the empty vector pLMP or the plasmid pshRNA-OCT1 and processed for Western Blot analysis using anti-hOCT1 antibody. Each lane contained 30 μg of total protein extract. Actin (ACT1) was used as the internal control. WT is the TOV2223G cells alone. Results are representative of three separate experiments. (B) Comparison of the concentration-dependent uptake of DNR into stable clones carrying either pLMP or pshRNA-OCT1. DNR uptake was monitored using the Fluoroskan Ascent microplate reader as above. Results are expressed as the mean ± S.D. from three separate experiments. (C) Epifluorescent microscopy reveals that OCT1 downregulation severely inhibits DNR uptake into TOV2223G cells. The stable clones of TOV2223G carrying either pLMP or the knockdown plasmid pshRNA-OCT1 were fixed with PFA, and processed for microscopy, using Axio Imager Z2 with Texas Red and DAPI-UV filters at 63× magnification. Images were processed with AxioVision software. Results are representative of three separate experiments. (D) Cell survival upon exposure to DNR. The TOV2223G cells and stable clones carrying either the empty vector pLMP or pshRNA-OCT1 were exposed to DNR (5 μM) for 72 h, and the viability of the cells was monitored using MTT assay. Results are expressed as the mean ± S.D. from two separate experiments.