X tremegene 9

293T cells were maintained in DMEM medium containing 10% FBS with antibiotics. For confocal imaging of live cells, cells were plated onto 35 mm glass bottom dishes (MatTek Corporation) 24 hours prior to transfection. GFP-PH-AKT and/or DsRed-PHLDA2 constructs (0.5–2 μg of DNA) were transfected into these cells using X-tremeGENE 9 at a 1:3 DNA: X-tremeGENE 9 ratio (Roche Molecular Biochemicals) and were visualized directly at 24 hours post-transfection. The plate was then placed on the heated stage of a Zeiss Axiovert 100TV inverted fluorescence microscope (Microcosm Inc., Columbia, MD) with the temperature maintained at 37°C. Cells were photographed using a Nikon G4 microscope (Sterling, MA) with a SPOT RT (Kodak, Rochester, NY) camera for digital imaging.

CHIP^+/+, CHIP^−/− and T246M CHIP mouse embryonic fibroblasts (MEFs) were cultured as previously described [28 (link)]. COS-7 and shCTRL and shCHIP HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Sigma) at 37 °C in an atmosphere of 5% CO₂. Cell transfections were performed using X-tremeGENE 9 (Roche) with the indicated plasmid DNA at a 1:3 DNA to X-tremeGENE 9 ratio.

HEK293T (CRL-11268) and HEK293 (CRL-1573) cells were purchased from American Type Culture Collection (ATCC) and were thus not authenticated. Cells were tested and confirmed to be mycoplasma free. Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Corning) supplemented with 10% fetal bovine serum in a tissue culture incubator at 37°C and 5% CO₂ in the absence of antibiotics. Transfections were performed with X-tremeGENE 9 (Roche), using a 3:1 ratio of X-tremeGENE 9 to plasmid DNA.

Human neuroblastoma SH-SY5Y cells were maintained as described.³⁷ (link) Cells were cultured to 40–50% confluence in 6-well or 12-well plates and transfected with plasmids using X-tremeGENE^TM 9 (Roche Life Science) according to the manufacturer’s instructions. We used non-tagged human tau 3R1N, 4R1N, haemagglutinin (HA)-tagged human tau 3R1N, 4R1N, and FLAG-tagged human tau 3R1N, 4R1N in the pCDNA3.1 vector. After transfection of plasmids, cells were incubated for 6–8 h, and pathogenic tau seeds (2 μl for 6-well plate or 1–2μl for 12-well plate) were introduced using MultiFectam (Promega) according to the manufacturer’s instructions. Transfected cells were incubated for 3 days.

Human cervical cancer (HeLa, SKGII, SKGIIIb, and ME180), ovarian cancer (SKOV3 and OKV18), and normal lung fibroblast (MRC5) cells were purchased from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan). All the cells lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 5–10% fetal bovine serum (Fujifilm WAKO Pure Chemical Corporation, Osaka, Japan) in a humidified incubator (37 °C and 5% CO₂). Wi-A and CAPE were dissolved in dimethyl sulfoxide (DMSO) (WAKO, Osaka, Japan) to make 5 mM stock and added to the complete cell culture medium, to obtain the working concentration as indicated. Cells grown at 60–70% confluency were treated with either Wi-A or CAPE or their combination for 48 h. For microscopic observations, cells were cultured in six-well plates, and we examined them live and in fixed states (as described below in immunocytochemistry section). Mortalin-targeting shRNA (shRNA-2166) was used for knockdown of mortalin as described previously [79 (link),80 (link)]. It was transfected into cells using X-tremeGENE^TM 9 (Roche, Basel, Switzerland), following the manufacturers’ protocol. After 24–48 h, the transfected cells were subjected to Western blot analysis.

Cells were plated into 12-well plates (1 × 10⁵ cells/well for HEK293 cells, 2 × 10⁵ cells/well for SW620 and NCI-H460 cells and 4 × 10⁵ cells/well for Panc10.05 cells) and incubated at 37 °C, 5% CO2 for 24 h before transfection with a 4:1 DNA ratio of pCMV6-Affimer-tGFP and FLAG-ERK plasmids using Lipofectamine 2000 (SW620, HEK293, NCI-H460) or X-tremeGENE 9 (Roche; Panc10.05). After 24 h cells were serum-starved for 1 h, HEK293 cells were then stimulated with 25 ng/ml EGF (Gibco) for 5 min (other cell lines were not stimulated). Cells were washed with ice-cold DPBS then incubated for 10 min on ice with NP-40 lysis buffer. Cleared lysates were incubated overnight at 4 °C with 20 μl anti-FLAG M2 magnetic beads (SigmaAldrich). The beads were washed 3× with TBS before protein elution by incubation at 95 °C for 5 min in SDS sample buffer. Levels of ERK and pERK were then analyzed by immunoblotting with anti-ERK antibody (1:2000, Abcam, ab184699) and phospho-ERK antibody (1:1000, Abcam, ab76299). Densitometry analysis used ImageJ software v.1.52 (NIH, Maryland).

For generation of HEK293 and HeLa reporter cell lines harboring the T2/CMV-EGFP.PB/PTK transposon cassette, 2.5 × 10⁵ cells/well were seeded 1 day prior to transfection in 6-well plates. Cells were co-transfected with 450 ng pT2/CMV-EGFP.PB/PTK and 50 ng of pCMV-SB100X using X-tremeGENE 9 (Roche, Basel, Switzerland) or TurboFect (Thermo Fisher Scientific, Waltham, MA, USA) for transfection of HEK293 or HeLa cells, respectively, according to the manufacturer’s instructions. One day after transfection, the cells were split into P10 dishes with appropriate dilutions, and the following day, the medium was changed to medium supplemented with 1 μg/mL puromycin (Sigma-Aldrich). Single puromycin-resistant colonies were isolated and expanded for further experiments. For generation of HeLa PB/Cas9-PuroTK cell lines, HeLa cells were transfected with 900 ng pPBT/EFS-Cas9-PuroTK and 100 ng pCMV-hyBPase using TurboFect, and single clones were isolated as described above.