Fig 1: shRNA-OCT1 downregulation in TOV2223G cells diminishes DNR uptake and confers resistance to the drug.(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.
Fig 2: High OCT1 mRNA levels are associated with better overall survival of high-grade serous epithelial ovarian cancer patients.Overall survival plot was generated online using the Kaplan-Meier Plotter based on signal intensity of the SLC22A1 probe (207201_s_at) in Affymetrix microarray gene expression data from high-grade serous epithelial ovarian cancer patients of The Cancer Genome Atlas. Auto select best cutoff was chosen in the analysis; cutoff value was 19 and expression range of the probe was 1–150. A total of 469 patients were available and samples were split in two groups (high and low) according to the cutoff value. The hazard ratio with 95% confidence intervals and log rank p value was calculated and significance was set at p < 0.05.
Fig 3: Expression of OCT1-EYFP, but not the OCT1-D474C variant, increases DNR uptake in HEK293T cells.The plasmid pOCT1-EYFP was used to create the OCT1-D474C mutation by site-directed mutagenesis. The plasmids pEYFP (empty), pOCT1-EYFP and pOCT1-D474C-EYFP were transiently transfected into HEK293T cells and exposed to DNR. Cells expressing EYFP were sorted to quantify the level of DNR uptake using FACS analysis using FL1 530/30 to detect EYFP fluorescence and FL4 670LP for DNR fluorescence. (A) Epifluorescent microscopy showing localization of the OCT1-EYFP fusion protein. Pictures were taken with Ziess Z2 fluorescent microcope using DAPI filter set and EYFP filter set. Results are representative of three separate experiments. (B) DNR uptake in HEK293T cells carrying either the plasmid pOCT1-EYFP, the variant, or the empty EYFP vector. Results are expressed as the mean ± S.D. from three separate experiments. (C) Comparison of the rhodamine uptake level in HEK293T cells carrying the empty EYFP vector, or either the plasmid pOCT1-EYFP or pOCT1-D474C-EYFP. Results are expressed as the mean ± S.D. from three separate experiments.
Supplier Page from R&D Systems, a Bio-Techne Brand for Human SLC22A1 (AAI26365) VersaClone cDNA
Available conjugates: Sizes Available: 10 ug