Lipofetamine rnaimax

Hoechst 33342, verapamil, glutaminase, L-asparaginase, and 3-Amino-1,2,4-triazole (ATZ), 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), hydroethidine, Rhodamine 123 were purchased from Sigma (St Louis, MO, USA). Rabbit monoclonal anti-Axin2 (D48G4), rabbit monoclonal anti-Survivin (71G4B7), rabbit polyclonal anti-phospho-β-catenin (Ser33/37/Thr41), rabbit monoclonal anti-phospho-Akt (Ser473) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal anti-c-Myc (9E10) was purchased from Santa Cruz (Santa Cruz, CA, USA). Rabbit polyclonal anti-CyclinD1 antibody was obtained from GeneTex (San Antonio, TX, USA). Mouse monoclonal anti-β-catenin (C47H1), rabbit monoclonal anti-Sox-2, rabbit monoclonal anti-ABCG2, and mouse monoclonal anti-β-actin antibodies were purchased from Abcam (Cambridge, UK). CM-DCFDA, Lipofetamine RNAiMAX and Opti-MEM were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).

A human fibroblast cell line (established from normal human skin, 27 years old, male, Asian) was kindly provided by Dr. Chung-Hsing Chang (School of Medicine, College of Medicine, China Medical University, Taichung, Taiwan), and the A549, GBM8401, and Huh-7 cell lines were obtained from the Bioresource Collection and Research Center. Cells were maintained in Dulbecco’s Modifed Eagle’s medium that was supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified atmosphere of 5% CO₂. To perform SRSF1 or SEDT2 knockdown, antisense oligonucleotide sequences of SRSF1, SEDT2, or a scrambled control (Supplementary Table S1) were transfected into cells using Lipofetamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol.

3T3‐L1 adipocytes were electroporated (Gene PulserXcell, Bio‐Rad) as previously described.19 For overexpression analysis, cells were electroporated with a phrGFP‐N1 expression vector (mock‐transfected cells) or a GFP‐Necc2 construct 18 and cultured for 48 hours prior to the experiments. For silencing studies, a specific shRNA for Necc2 silencing (5′‐GGAGGAGATAAGATTTAAA‐3′) was cloned using the BgIII and HindIII sites in front of the H1‐RNA promoter of the pEGFP‐RNAi plasmid as described earlier.18 Cells expressing pEGFP‐shRNA (control shRNA) or pEGFP‐Necc2‐shRNA plasmid (NECC2 shRNA) were kept in culture for 72 hours before performing the experiments.
For colocalization analysis of NECC2 and cavin1, cells were transfected with Lipofectamine 2000 (Invitrogene, Barcelona, Spain) and the expression vector coding for GFP‐Necc2, cultured for 48 hours and then immunostained for cavin1. For Cav1 silencing studies, 3T3‐L1 adipocytes were transfected with Lipofetamine RNAiMAX (Invitrogen) and Cav1 siRNA (Dharmacon, Lafayette, CO, USA) and cultured for 72 hours before NECC2 immunostaining.

Plasmid EF.hHES1.Ubc.GFP and plasmid EF.deltaBHES1.Ubc.GFP were acquired from Linzhao Cheng’s lab [24 (link)] via addgene Inc. (Cambridge, MA). The Flag-Hes1 was generated by digesting EF.hHES1.Ubc.GFP with BamHI and XhoI, and inserting the digested plasmid into the multiple cloning sites of the pcDNA4-TAG vector (Invitrogen, Carlsbad, CA). Stattic was obtained from Sigma-Aldrich (St. Louis, MO). Control siRNA and Hes1 siRNA were obtained from Qiagen (Venlo, Netherlands). Lipofetamine RNAiMAX (Invitrogen) was used as an siRNA transfection reagent. The protocol was done according to the manufacturer’s instructions at a final concentration of 10nM.