Rnaimax transfection reagent

Cultured cells were transfected with plasmid DNAs using RNAi Max transfection reagent (Invitrogen) according to the manufacturer’s protocol. At 48 h following transfection, cells were harvested for immunofluorescent staining and Western blotting. For caspase-4 knockdown experiment, the cultured human neural SH-SY5Y cells were transiently transfected with caspase-4 siRNA (Gene Pham Co. sequence GUGUAGAUGUAGAAGAGAAtt or AAGUGGCCUCUUCACAGUCAUtt) or control siRNA (scrambled sequence) using RNAi Max transfection reagent (Invitrogen) according to the manufacturer’s protocol. At 48 h following transfection, cells were harvested for immunofluorescent staining and Western blotting.

On the day of the transfection, frozen imaging assay-ready plates were thawed, equilibrated at room temperature, and centrifuged at 500 g for 5 min. Then, 20 µl of OPTIMEM (31985088, Thermo Fisher Scientific) containing 50 nl of RNAiMax transfection reagent (13778075, Thermo Fisher Scientific) was added to each well of the imaging plate and incubated for 30 min at room temperature. Next, 20 µl of trypsinized and resuspended HeLa^{YFP–CENP-A-high} cells in DMEM containing 20% FCS was added at a concentration of 1500 cells/well. Plates were incubated at room temperature for 30 min and then at 37°C for 72 h. The final concentration of each siRNA was 20 nM. At 72 h post transfection of the siRNA, cells were fixed in paraformaldehyde (PFA; 28908, Thermo Fisher Scientific) for 15 min at room temperature by adding 40 µl per well of 8% PFA in PBS directly to the medium using a Bluewasher plate washer/dispenser (BlueCatBio). The same instrument was used to wash plates in 50 µl/well of PBS and to stain with 50 µl/well of DAPI (0.5 µg/ml). Plates were stored at 4°C until imaged. Two independent biological replicates of the screen were performed.

Cells were transfected with STK19 siRNA (siGENOME SMARTpool, Dharmacon M-005378-02) or non-targeting control (siGENOME Non-Targeting siRNA Pool #2, Dharmacon D-001206-14) using RNAiMAX transfection reagent (Thermo Fisher Scientific 13778030) following manufacturer instruction. Briefly, cells were seeded at 40% confluency in 6 well plates and transfected with 50nmol (FlpIn T-Rex HEK293) or 15nmol (FlpIn T-Rex HeLa, SK-MEL-2 and UACC_62) and knock down efficiency assayed 72 h after transfection. For transfection with EPr mini-gene constructs and empty vector controls, cells at 50% confluency were seeded in 6 well plates and transfected using Lipofectamine 2000 (ThermoFisher Scientific 11668027) following manufacturer instructions, and analyzed 24 h later.

Primary Sertoli cell obtained from testes of 3 weeks old mice testes were cultured for 3 days, and then subjected to transfection with 100 nM non-targeting or Senp3-specific siRNA duplexes for 24 hours by RNAi MAX Transfection Reagent according to the manual (Thermo Scientific, Waltham, MA). After transfection, the reaction mixture was removed and replaced with fresh F12/DMEM, and cells were cultured for another 24 h before termination for immunofluorescent analysis (IF) or for RNA extraction and lysate preparation. The Senp3-specific siRNA (sc-45718; Santa Cruz, CA) and non-targeting siRNA (sc-37007; Santa Cruz, CA) control duplexes were purchased from Santa Cruz and the sequences are shown in Supplementary Table 1.

C1orf116 siRNA (ThermoFisher, cat#: 4,392,420) with RNAiMAX transfection reagent (ThermoFisher) was used for siRNA transfections. Some alterations were made to manufacturer’s recommended protocol. Cells were seeded at a density result in 50% confluency the following day. Using a 6 well plate, 9 ul of RNAiMAX reagent and 3 ul (30 pmol) of siRNA (each diluted in 150 ul of Opti-MEM media) was added to each well the day after seeding. 72 h later RNA was isolated (Qiagen, Rneasy mini kit) from plates and gene expression was analyzed.

Mimics and inhibitors of miR-122 and miR-378 were used to overexpress or knockdown miRNA in HepaRG cells. Cells were grown in 24 well plate (see cell culture) and transfected with 5 nM mimic, 50 nM or 100 nM inhibitor using RNAiMAX Transfection Reagent (ThermoFisher Scientific, Carlsbad, CA). At 24 h, the transfection medium was replaced with HepaRG cells Tox Working Medium. At 72 h, cells were cultured in glutathione free media. APAP (20 mM) was added for 12 h and cells were harvested at 96 h post transfection for protein analysis. Transfection efficiency of 65–89% was observed with 5–50 nM concentration of AllStars Hs Cell Death Control siRNA. Preliminary studies found no interference in protein expression with negative control AllStars siRNA.

In the study, we used commonly used commercial human breast cancer cell lines. BT474 and T47D were maintained in RPMI (BT474, T47D) or DMEM (MCF7, Hs578T, CAL51, MDA-MB-231) containing 10% FBS, 1.5 g·L⁻¹ of NaHCO₃, 10 mM HEPES, 1× penicillin/streptomycin, 1 mM sodium pyruvate, 5 mM glucose, and 4 mM glutamine. Cell lines expressing estrogen receptor (MCF7, BT474, T47D) were maintained in phenol-red-free media supplemented with 10 nM 17-β-estradiol. Transfection of vector DNA was performed using Lipofectamine2000 (Thermofisher Scientific (Waltham, MA, USA)) or GeneGet transfection reagent (SignaGene) according to the manufacturer´s protocols. siRNA was transfected using RNAiMAX transfection reagent (Thermofisher Scientific). Knockdown of ADHFE1 was performed using a mixture of three predesigned Stealth siRNAs purchased from ThermoFisher Scientific (HSS134689, HSS13469, and HSS175147). IDH2 WT fused with a Flag-tag was a kind gift from the laboratory of Dr. Denu [28 (link)]. Vectors encoding IDH2 R140Q, IDH2 WT, and IDH2 R172K were kind gifts of Craig Thompson´s Laboratory. Vector encoding GAC K320A was a kind gift of Dr. Sandra Martha Gomes Dias [23 (link)].