Cell culture, reagents and antibodies
KINGS-1 cells (GIII astrocytoma, HSRRB) were maintained in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin and 50 mg/ml streptomycin. U-87MG (Glioblastoma, ATCC) cells were maintained in Eagle's minimal essential medium (E-MEM) supplemented with 10% FBS, 50 units/ml penicillin and 50 mg/ml streptomycin. HEK293T cells, MDA-MB-231cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. A7 cells were provided from Dr. Thomas P. Stossel (Harvard University). A7 cells were maintained in Minimum Essential Medium Eagle supplemented with 8% newborn calf serum, 2% fetal calf serum, 50 units/ml penicillin and 50 mg /ml streptomycin. Reagents, antibodies, siRNA used in this study were listed in supplementally table 1.
Transfection
KINGS-1 cells were transfected with plasmid DNA for 24 hours using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. HEK293T cells was transfected with plasmid DNA for 24 hours using Poly-Ethylene-Imine (PEI, polyscience). KINGS-1, U-87MG cells were transfected with FilGAP siRNA for 48 or 72 hours using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. The cells were maintained at 37°C with 5% CO₂ during the treatments.
Immunoblotting
Total cellular proteins were harvested using RIPA buffer [20 mM Tris‐HCl (pH7.5), 120 mM NaCl, 1% TritonX-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mM MgCl2, 1 mM EDTA, 10 mM NaF, 10 mM b-glycerophosphate, Protease inhibitor Cocktail and 1 mM DTT]. Cell lysates were separated by SDS‐PAGE and transferred to the membrane for fluorescence (Millipore). The membrane was blocked with Intercept (PBS) Blocking Buffer (LI-COR) and incubated with primary antibodies. The primary antibodies were detected with secondary antibodies and FUSION SOLO S (Vilber Loumat).
GST pulldown assay
HEK293T cells were washed with phosphate-buffered saline (PBS), suspended with CHAPS buffer (50mM Tris-HCl [pH 7.5], 0.1M NaCl, 2mM MgCl2, 0.1mM EDTA [pH 8.0], 0.3% CHAPS, Protease inhibitor Cocktail (SIGMA), and 1mM DTT). The cells were disrupted and the cell lysates were prepared by centrifugation for at 15,000 rpm for 5 min at 4°C. The supernatant fluid was incubated with GST-FilGAP protein coupled with glutathione-Sepharose beads for 60 min at 4 °C. The beads were washed three times with CHAPS buffer and bound Raptor, Rictor or mTOR were detected by SDS-PAGE followed by Western blot using corresponding antibody.
Immunoprecipitation
HEK293T cells were washed with PBS, suspended with CHAPS buffer. Cell lysates were precleared and supernatant fluid was incubated with 20µl of monoclonal anti-Flag agarose beads for 1hour at 4℃. Immunoprecipitates were washed three times with CHAPS buffer and bound proteins were detected by SDS-PAGE followed by Western blot.
Plasmids
The FilGAP siRNA KD#2-resistant construct (rKD#2) was generated by introducing point mutations at nucleotide positions 771, 777, 780, 786, and 792 of the FilGAP coding sequence using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Sin1 was cloned from HEK293T cells by RT-PCR, and subcloned into pCMV5-Flag, pCMV5-Myc and pMAL-c2X. FilGAP, Raptor and Sin1 deletion mutants were generated by PCR. pEF-Bos-Flag-Rictor, pRK5Myc-Raptor was gifted from T Sato (Aichi Cancer Research Institute).
RT-PCR
cDNA was synthesized from 0.5 mg of total RNA by ReverTraAce (TOYOBO) and amplification was carried out using specific forward primers for ARHGAP24 gene as follows 45: variant 1 primer located: 5’- CTG CAA TGA AGA GAA CCC AG-3’, variant 2: 5’- ATG CCT GAA GAC CGG AAT TC-3’, variant 3: 5’- CAG TGG ACA GTT AAA CAA GAG-3’, and variant 4: 5’- GTCACTGACCACTGAAGTGT-3’. Common reverse primer located 5’-CAT AAC GAA CAG TAT CCT CCA G-3’. β-actin was used as a loading control.
Spheroid culture
For spheroid generation, 200 ml/well of control or FilGAP-depleted KINGS-1 and U-87MG cells suspensions at optimized densities (0.5×10⁴ cells/mL) were dispensed into 96-well round-bottomed ultra-low attachment surface plates (Corning or Greiner). Spheroids were incubated for 9 days at 37°C, 5% CO₂ with 50% medium exchange every 3 days. Images of the spheroids were acquired every 3 days and their diameters were measured with Image J.