Bay36 7620

Riluzole hydrochloride (Tocris Bioscience, Bristol, UK) and BAY 36-7620 (Tocris Bioscience, Bristol, UK) were dissolved in DMSO. GRM1 primary antibody is specific to the C-terminal region of human GRM1α and was used at a dilution of 1:2000 for Western blotting (catalog number 36350002; Novus Biologicals, Littleton, CO). Additional primary antibodies for Western blotting or immunofluorescence include monoclonal anti-ß-actin clone AC-15 (Sigma Aldrich, St. Louis, MO); phospho-cdc2 (Tyr15), cyclin B1, phospho-histone H2AX (Ser139) (Cell Signaling, Danvers, MA). Primary antibodies for flow cytometry include phospho-histone H3 (Ser10) rabbit monoclonal (Alexa Fluor^® 488 conjugate), histone H3 rabbit monoclonal (Alexa Fluor^® 647 conjugate), rabbit monoclonal IgG isotype control (Alexa Fluor^® 488 conjugate), rabbit monoclonal IgG isotype control (Alexa Fluor^® 647 conjugate) (Cell Signaling, Danvers, MA).

Rabbit anti-mGluR1 and β-TRCP antibodies as well as mouse anti-BrdU antibodies were from Cell Signaling, Beverly, MA. Mouse anti-glutaminase and rabbit anti-mGluR1 and -TATA binding protein (TBP) antibodies were from Abcam, Cambridge, MA. Mouse anti-tubulin and β-actin antibodies were from Sigma, St. Louis, MA. Mouse anti-c-Myc (9E10) and REST antibodies were from Santa Cruz, Santa Cruz, CA. Rat anti-Kaposin A/C and mouse anti-polyubiquitin antibodies were from Millipore, Temecula, CA. Mouse anti-ORF73 antibodies were generated in Dr. Chandran's laboratory. Anti-rabbit and anti-mouse antibodies linked to horseradish peroxidase were from KPL Inc., Gaithersburg, Md. Alexa 488 and 594 conjugated secondary antibodies were from Invitrogen. Protein A and G–Sepharose CL-4B beads were from Amersham Pharmacia Biotech, Piscataway, NJ. Lambda phosphatase (λPPase), and L-DON (6-diazo-5-oxo-norleucine) were from Santa Cruz. Riluzole, A841720 and Bay 36-7620 were from Tocris Bioscience, Minneapolis, MN.

The MCF10A series of cell lines were originally developed at the Michigan Cancer Foundation (the institutional precursor to the Karmanos Cancer Institute) and are available to us from original stocks at low passage [28] (link), [31] (link)–[33] (link). MCF10A series cell lines were cultured in DMEM/F12 (1∶1) media (Life Technologies, Carlsbad, CA) supplemented with 5% horse serum, 10 µg/ml insulin, 20 µg/ml EGF, 0.5 µg/ml hydrocortisone and 100 ng/ml cholera toxin plus antibiotics at 37°C, 5% CO₂. MCF10A and MCF10AT1 transduced with pLenti-GRM1 or control LacZ were grown with 5 to 8 µg/ml blasticidin (Life Technologies, Carlsbad, CA); MCF10DCIS.com and MCF10CA1d transduced with lentiviral GIPZ shGRM1 or non-silencing control were maintained in 2 µg/ml puromycin (Life Technologies, Carlsbad, CA). The noncompetitive mGluR1 inhibitor BAY36-7620 [(3aS,6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopental [c]-furan-1-on], competitive mGluR1 inhibitor LY367385 [(S)-(+)-α-Amino-4-carboxy-2-methylbenzeneacetic acid], and mGluR1 agonist L-quisqualate [(L)-(+)-a-amino-3,5-dioxo-1,2,4-oxadiazolidine-2-propanoic acid] were purchased from Tocris Bioscience (Ellisville, MO, USA).