Phosphorylated ribosomal protein s6

Myoblasts were grown on 6-well plates as described, washed in PBS, lysed, and scraped in 150 mL RIPA (Pierce/Thermo Scientific Waltham, MA USA, 89900) supplemented with PhosSTOP (Roche Penzberg, Germany, 04 906 837 001) and 1% protease inhibitor cocktail (Sigma P8340). Cells were lysed at 4°C for 20 minutes, centrifuged at 12,000g for 15 min, and the pellet was discarded. Protein concentrations were measured by BIO-RAD DC assay, and 5–10 mg of protein was run. Primary antibodies and dilutions: DUX4 E55 1:1000 (AbCam, Cambridge, UK, ab124699), Vinculin 1:10,000 (Sigma V9131), phosphorylated ribosomal protein S6 1:1000 (Cell Signaling Danvers, MA USA, 4858S), ribosomal protein S6 1:1000 (Cell Signaling 2317S), phosphorylated AKT 308 1:1000 (Cell Signaling 13038P), phosphorylated AKT 473 1:1000 (Cell Signaling 4060S), Pan-AKT 1:1000 (Cell Signaling 4691P), ULK1 1:1000 (Cell Signaling 8054T/S), and LC3A/B 1:1000 (Cell Signaling 12741T). Secondary antibodies and dilutions: anti-rabbit and anti-mouse IgG-HRP 1:2000 (Cell Signaling 7074P2 and 7076S). ECL reagents: Clarity Western ECL Substrate (BIO-RAD, Hercules, CA 102031761) and Clarity MAX Western ECL Substrate (BIO-RAD 76SF121P). Blots were visualized on a BIO-RAD ChemiDoc. Uncropped versions of all western blots are presented in the supplementary materials.

1-(3-Chloro-4-((trifluoromethyl)thio)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea (FND-4b) was synthesized and characterized as previously described [28 (link)]. Roswell Park Memorial Institute (RPMI) 1640 Medium and Eagle’s Minimum Essential Medium (EMEM) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified Eagle Medium (DMEM) was from Corning (Corning, NY). Human breast cancer stem cell complete growth medium was from Celprogen (Torrance, CA). MEM non-essential amino acid solution (100x), sodium pyruvate solution (100 mM), insulin solution (10 mg/mL), penicillin-streptomycin (100x) (P/S), and fetal bovine serum (FBS) were from Sigma-Aldrich. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) was from Abcam (Cambridge, MA). Antibodies for pAMPKα (Thr172), total AMPKα, phosphorylated acetyl-CoA carboxylase (ACC), total ACC, phosphorylated ribosomal protein S6, total S6, and PARP were from Cell Signaling Technology (CST; Danvers, MA). The cyclin D1 antibody was from Abcam. The beta-actin antibody was from Sigma-Aldrich. The secondary antibodies to rabbit and mouse were from Santa-Cruz. All relevant antibody information is in Table 1. The Sulforhodamine B (SRB) Cytotoxicity Assay was from G-Biosciences (St. Louis, MO). The Cell Death Detection ELISA^PLUS assay was from Sigma-Aldrich.

Mouse tumor tissues were kept in 4% paraformaldehyde at 4 °C. The specimens were then processed, embedded in paraffin and sectioned at 5 μm. Heat-induced antigen retrieval was performed in citrate buffer using a pressure cooker. Sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies used were phosphorylated S6 ribosomal protein (Cell Signaling Technology, Danvers, MA, USA), S6 ribosomal protein (Cell Signaling Technology), Ki67 (Spring Bioscience Corporation, Pleasanton, CA, USA), GFAP (Cell Signaling Technology), Sox2 (Millipore Sigma, Burlington, MA, USA), DCX (Millipore). Sections were then incubated with FITC- or TRICT-conjugated IgG (for 1 h at room temperature), followed by DAPI staining (Vector Laboratories, Burlingame, CA, USA). Images were acquired with Olympus BX41 microscope. Six random fields from three to five different tissues in each group were quantified using ImageJ software.