Hiperfect transfection reagent transfection protocol

For silencing experiments, TRPML-1 (siTRPML-1) and siCONTROL non-targeting siRNA (siGLO, used as negative control) FlexiTube siRNA were purchased from Qiagen (Milan, Italy). For gene silencing experiments, T98 and U251 cell lines were plated at the density of 1.2 × 10⁵/mL and siTRPML-1 or siGLO (150 ng for T98, 75 ng for U251) was added to the wells, following the HiPerfect transfection reagent transfection protocol (Qiagen). No differences were observed comparing siGLO transfected with untransfected cells.
For overexpression experiments, glioma cells were plated at a density of 1.2 × 10⁵/mL. After overnight incubation, transfections were achieved with 7.5 μL/mL of the reagent TransIT-X2 (Mirus MIR-6003, OriGene, Rockville, MD, USA) and 2.5 μg/mL of pCMV-pTRPML-1 or pCMV empty (pCMV) vectors according to the manufacturer’s instructions (Origene, Castenaso, Italy). No differences were observed comparing pCMV transfected with untransfected cells.

For silencing experiments, the previously characterized mesenchymal GSC#83 cells (1.2 × 10⁵/mL) [22 (link)] were transfected with TRPML2 (siTRPML2, 150 ng) and siCONTROL non-targeting siRNA (siGLO, used as negative control, 150 ng) Flexi Tube siRNA (Qiagen, Milan, Italy), following the HiPerfect transfection reagent transfection protocol (Qiagen). There are no differences between siGLO transfected and untransfected cells.
For overexpression experiments, the proneural GSC#1 cells (1.5 × 10⁵/mL) [22 (link)] were transfected with 10 μL/well of Roti-Fect (Carl Roth GmbH, Karlsruhe, Germany) and 2 μg/well of pCMV3-MCOLN2-t1 (pCMV-TRPML2) (Sino Biological, Wayne, PA, USA) or pCMV3 empty (pCMV) vectors according to the manufacturer’s instructions. There are no differences between pCMV transfected and untransfected cells.

For silencing experiments, TRPML2 (siTRPML2) and siCONTROL non-targeting siRNA (siGLO, used as negative control) FlexiTube siRNA were purchased from Qiagen (Milan, Italy). For gene silencing experiments, T98 and U251 cell lines were plated at a density of 1.2 × 10⁵/mL and siTRPML2 or siGLO (150 ng) was added to the wells, following the HiPerfect transfection reagent transfection protocol (Qiagen, Milan, Italy). No differences were detected comparing siGLO control cells with untransfected cells.
For overexpression experiments, 1.5 × 10⁵/mL T98 and U251 cells were plated. After overnight incubation, transfections were achieved with 10 µL/well of Roti-Fect (Carl Roth GmbH, Karlsruhe, Germany) and 2 µg/well of pCMV3-MCOLN2-t1 (pCMV-TRPML2) (Sino Biological, Wayne, PA, USA) or pCMV3 empty (pCMV) vectors according to the manufacturer’s instructions. No differences were observed comparing pCMV transfected with untransfected cells.