Jetpei cationic polymer transfection reagent

MCF7 and HeLa cells were transfected in 50% confluent 24-well plates using jetPEI cationic polymer transfection reagent (Polyplus Transfection, Illkirch, France). Co-transfections with an E2F1 expression vector were preceded by 48 hr. serum starvation (with 0.1% FBS containing medium). Where indicated PTEN, TPIPα or TPIPβ pEGFP-C2 expression constructs were added (generous gift of Nicholas Leslie, University of Dundee).
Firefly and Renilla luciferase activities were quantitated sequentially using the Dual Luciferase Assay system (Promega) in a MiniLumat LB 9506 luminometer (EG&G Berthold, Germany). Where indicated, the pRL-TK, a Renilla luciferase plasmid driven by the HSV-thymidine kinase (TK) promoter (Promega), was used to normalize for transfection efficiencies by calculating the relative firefly/Renilla luciferase activities (RLA). Results of duplicates were averaged, and corrected for plasmid size differences to reflect equimolar measurements. Statistical analysis was performed using student's t-test.

The antibodies against LPIN1 (used at a dilution ratio of 1:1000), phosphorylated-COT (1:500), phosphorylated-JNK1/2 (1:500), and phosphorylated-c-Jun (1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against MYC (1:5000) and β-actin (1:10,000) were from Sigma-Aldrich (St. Louis, MO, USA). ST2 (1:1000), COT (1:1000), JNK1 (1:1000), and JNK2 (1:1000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-V5 antibodies were obtained from Invitrogen (Carlsbad, CA, USA). Recombinant human IL-33 was obtained from R&D Systems (Minneapolis, MN, USA). The COT inhibitor TKI, PI3K/Akt inhibitor LY294002, STAT3 inhibitor WP1066, MEK inhibitor PD98059, and JNK inhibitor SP600125 were purchased from Calbiochem-Novabiochem (San Diego, CA, USA). The Ez-Chip kit and polyvinylidene difluoride (PVDF) membranes were purchased from Millipore (St. Louis, MO, USA). The ImProm-II™ Reverse Transcription System was purchased from Promega (Madison, WI, USA). The jetPEI^® cationic polymer transfection reagent was purchased from Polyplus-transfection (NY, USA). Lipofectamine™ LTX Reagent with PLUS™ transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA).