Anti phospho mtor

To induce cellular stress, sodium arsenite (arsenite, NaAsO₂; Sigma-Aldrich) and potassium tellurite (tellurite, K₂TeO₃ kindly gifted by Dr. José Manuel Pérez-Donoso, UNAB, Chile) were used. Cycloheximide and Puromycin were obtained from Sigma Aldrich. The antibodies used in this work included the following: anti-phospho-eIF2α (rabbit polyclonal) and anti-GAPDH (mouse monoclonal) from Abcam; anti-eIF2α (mouse monoclonal), anti-phospho-S6K1 (rabbit monoclonal), anti-phospho-S6 (rabbit monoclonal), anti-phospho-4EBP1 (rabbit monoclonal) from Cell Signaling Technology; anti-Puromycin (mouse monoclonal) from Millipore; anti-DCP1a (mouse monoclonal), anti-eIF3b (mouse monoclonal), anti-TIAR (goat polyclonal), anti-DDX3 (rabbit polyclonal), anti-phospho-mTOR (mouse monoclonal), anti-mTOR (mouse monoclonal), anti-S6K1 (mouse monoclonal), anti-S6 (mouse monoclonal), anti-4EBP1 (mouse monoclonal) from Santa Cruz Biotechnology and anti-SC35 (mouse monoclonal) kindly provided by Dr. Verónica Noches, PUC, Chile. Secondary antibodies used for immunofluorescence included the following: Alexa Fluor 594 donkey anti-mouse, Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 647 donkey anti-mouse or anti-goat from Life Technologies; and secondary antibodies for western blot (horseradish peroxidase-conjugated) anti-mouse and anti-rabbit were purchased from Jackson Immunoresearch.

The human TC cell lines TPC1 and 8505C, derived from PTC and ATC, respectively, were cultured with RPMI-1640 (Gibco, Thermo Scientific, MA, USA) medium containing 10% fetal bovine serum (FBS) (Gibco, Thermo Scientific, MA, USA) and 1% penicillin at 37 °C with 5% CO₂. LY294002 (Biogems International, CA, USA), Lipofectamine 2000 (Invitrogen, CA, USA), Nile red (Santa Cruz Biotechnology, Texas, USA), G418 (GenePharma, Shanghai, China), puromycin (Gibco, Thermo Scientific, MA, USA), and polybrene (GenePharma, Shanghai, China) were also used in this study. The antibodies used were anti-PC (Santa Cruz Biotechnology, TX, USA), anti-SREBP1c (Affinity Biosciences, OH, USA), anti-FASN (Abcepta, CA, USA), anti-phospho-mTOR (Santa Cruz Biotechnology, TX, USA), anti-mTOR (Proteintech Group, IL, USA), and anti-phospho-Akt (Cell Signaling Technology, MA, USA). Antibodies against ACC1 (Proteintech Group, IL, USA), ACLY (Epigentek, NY, USA), E-cadherin (Affinity Biosciences, OH, USA), Vimentin (Affinity Biosciences, OH, USA), snail1 (Proteintech Group, IL, USA), ZEB2 (Proteintech Group, IL, USA), GAPDH (Beyotime Biotechnology, Shanghai, China), and α-Tubulin (Beyotime Biotechnology, Shanghai, China) were also used in this study.