Metafectene si pro transfection protocol

siGENOME SMARTpools for Beclin 1 (siBeclin 1), siCONTROL nontargeting siRNA (siGLO) used as negative control were purchased from Thermo Scientific-Dharmacon (CO, USA). For gene silencing experiments, 5637 and T24 cells were plated in a six well plate at the density of 3 × 10⁴/ml and 160 pmol of siBeclin 1 or siGLO was added to the wells for 48 h, following the METAFECTENE SI PRO transfection protocol (Biontex Laboratories, CA, USA).
To knockdown PTCH2, the GeneSolution siRNA for PTCH2 (siPTCH2), negative control siRNA duplex (siGLO) and the HiPerfect Transfection reagent (Qiagen, CA, USA) were used following the Fast-forward Transfection protocol. Briefly 5637 and T24 cells were plated in a twenty-four well plate at the density of 5 × 10⁴/ml and siPTCH2 or siGLO at the concentration of 5nM were added to the wells for 48 h. Then silenced cells were detached, counted and plated for MTT, western blot and FACS analyses.

siGENOME SMARTpools for TRPML-2 (siTRPML-2), siCONTROL non-targeting siRNA (siGLO) used as negative control were purchased from Thermo Scientific-Dharmacon (Lafayette, CO, USA). For gene silencing experiments, T98 and U251 cell lines were plated at the density of 3 × 10⁴/ml and 160 pmol of siTRPML-2 or siGLO was added to the wells, following the METAFECTENE SI PRO transfection protocol (Biontex Laboratories, San Diego, CA, USA).