Colon cancer LoVo cells sensitive (LoVo-S) or resistant to DXR (LoVo-R) (kindly donated by Dr. P. Perego, Istituto Nazionale Tumori, Milano) were cultured in DMEM containing 10% fetal bovine serum and 2 mM glutamine at 37 °C and 5% CO2.
To obtain a transient downregulation of TRPM6 and 7, we utilized the stealth siRNAs developed by Qiagen37 (link). siRNAs were transfected into 2 × 104/cm2 cells using HiPerFect Transfection Reagent (Qiagen). Non-silencing, scrambled sequences were used as controls (CTR).
To evaluate sensitivity to DXR, cell viability was assessed by MTT assay. Briefly, cells were seeded in 96 well/plates. Where indicated, 16 h prior to DXR treatment, LoVo-S were silenced for TRPM7 or TRPM6, or exposed to 2-aminoethoxydiphenyl borate (2-APB, 50 μM), while LoVo-R were treated with calpeptin (5 μg/ml). After 48 h of DXR treatment (1 μg/ml), culture medium was replaced with medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, 0.5 mg/ml) (Sigma, Oakville, Ontario, Canada). At the end of the experiment, media were removed and formazan crystals generated by cellular reductase activity were dissolved in DMSO:ethanol (1:1) and quantified by absorbance readings at 575 nm. The experiment was repeated three times in quadruplicates. Percentage cell viability was calculated from the absorbance measured in DXR-treated vs. untreated cells for each experimental condition.
To assess DXR retention, cells were loaded with 10 μM DXR for 2 h and fixed either immediately (loading control) or 30 min after washing (efflux). Nuclear fluorescence was visualized by confocal microscopy and quantified as the mean nuclear fluorescence measured in efflux conditions vs. loading control.
To obtain a transient downregulation of TRPM6 and 7, we utilized the stealth siRNAs developed by Qiagen37 (link). siRNAs were transfected into 2 × 104/cm2 cells using HiPerFect Transfection Reagent (Qiagen). Non-silencing, scrambled sequences were used as controls (CTR).
To evaluate sensitivity to DXR, cell viability was assessed by MTT assay. Briefly, cells were seeded in 96 well/plates. Where indicated, 16 h prior to DXR treatment, LoVo-S were silenced for TRPM7 or TRPM6, or exposed to 2-aminoethoxydiphenyl borate (2-APB, 50 μM), while LoVo-R were treated with calpeptin (5 μg/ml). After 48 h of DXR treatment (1 μg/ml), culture medium was replaced with medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, 0.5 mg/ml) (Sigma, Oakville, Ontario, Canada). At the end of the experiment, media were removed and formazan crystals generated by cellular reductase activity were dissolved in DMSO:ethanol (1:1) and quantified by absorbance readings at 575 nm. The experiment was repeated three times in quadruplicates. Percentage cell viability was calculated from the absorbance measured in DXR-treated vs. untreated cells for each experimental condition.
To assess DXR retention, cells were loaded with 10 μM DXR for 2 h and fixed either immediately (loading control) or 30 min after washing (efflux). Nuclear fluorescence was visualized by confocal microscopy and quantified as the mean nuclear fluorescence measured in efflux conditions vs. loading control.
Free full text: Click here
