ON-TARGETplus Human ITPR3 (3710) siRNA - SMARTpool

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	Human ovarian cancer cell line A2780 (Sigma-Aldrich, 93112519) were cultured in Minimal Essential Medium of Dulbecco (DMEM; Sigma, USA) or RPMI medium (Sigma, USA) with a high glucose (4.5 g L−1) and L-glutamine (300 μg mL−1), supplemented with 10% fetal bovine serum (Sigma, USA), penicillin (Calbiochem, USA; 100 U mL−1), and streptomycin (Calbiochem, USA; 100 μg mL−1). Cells were cultured in a water-saturated atmosphere at 37 °C and 5% CO2. Cells were grown in 6-well plates in RPMI with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in Hudecova et al.6. ON-TARGET plus SMART pool human ITPR3 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments. Based on the previous calibration, silencing was performed for 48 h. After the first 24 h of silencing, apoptosis inducer kit (AIK) was applied for an additional 24 h. Finally, all groups of cells were harvested and used in further experiments.The efficiency of the IP3R1 and IP3R3 silencing was measured by Western blot analysis, as described below.

Protocol tips

Human ovarian cancer cell line A2780 (Sigma-Aldrich, 93112519) were cultured in Minimal Essential Medium of Dulbecco (DMEM; Sigma, USA) or RPMI medium (Sigma, USA) with a high glucose (4.5 g L−1) and L-glutamine (300 μg mL−1), supplemented with 10% fetal bovine serum (Sigma, USA), penicillin (Calbiochem, USA; 100 U mL−1), and streptomycin (Calbiochem, USA; 100 μg mL−1). Cells were cultured in a water-saturated atmosphere at 37 °C and 5% CO2. Cells were grown in 6-well plates in RPMI with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in Hudecova et al.6. ON-TARGET plus SMART pool human ITPR3 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments. Based on the previous calibration, silencing was performed for 48 h. After the first 24 h of silencing, apoptosis inducer kit (AIK) was applied for an additional 24 h. Finally, all groups of cells were harvested and used in further experiments.The efficiency of the IP3R1 and IP3R3 silencing was measured by Western blot analysis, as described below.

Publication protocol

Human ovarian cancer cell line A2780 (Sigma-Aldrich, 93112519) were cultured in Minimal Essential Medium of Dulbecco (DMEM; Sigma, USA) or RPMI medium (Sigma, USA) with a high glucose (4.5 g L−1) and L-glutamine (300 μg mL−1), supplemented with 10% fetal bovine serum (Sigma, USA), penicillin (Calbiochem, USA; 100 U mL−1), and streptomycin (Calbiochem, USA; 100 μg mL−1). Cells were cultured in a water-saturated atmosphere at 37 °C and 5% CO2. Cells were grown in 6-well plates in RPMI with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in Hudecova et al.6. ON-TARGET plus SMART pool human ITPR3 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments. Based on the previous calibration, silencing was performed for 48 h. After the first 24 h of silencing, apoptosis inducer kit (AIK) was applied for an additional 24 h. Finally, all groups of cells were harvested and used in further experiments.The efficiency of the IP3R1 and IP3R3 silencing was measured by Western blot analysis, as described below.

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