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Calcusyn Software Manual3/8/2021 All cells were grown in 5% CO 2 at 37°C in a humidified tissue culture incubator.. Cells were routinely tested for mycoplasma contamination using a MycoAlert mycoplasma detection kit (Lonza, Rockland, ME) and were found to be negative. Autosomal short tandem repeat typing was conducted across the 13 core STRs in CODIS and referenced against allelic peaks in cell lines of previously confirmed genotype.. All cell lines were verified as authentic Proliferation assay Cells were seeded in 96-well plates and incubated overnight.. Cultures were exposed to various concentrations of CFZ or CDDP for the specified treatment intervals.. Proliferation of adherent cells was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.. These cells represent different pathological subtypes (squamous, adenocarcinoma, carcinoma) with SCLC cells derived from both metastatic lesions (SHP77) and a primary tumor (DMS114). Results CFZ treatment inhibited both the constitutive proteasome and the immunoproteasome in lung cancer cell lines. The Mac Hack ![]()
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Antibodies against microtubule-associated protein-1 light chain-3B (LC3B) were obtained from Sigma-Aldrich.. Proliferation of suspension cells was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.. A variety of molecular characteristics are also represented including wild-type p53 (H549, H460), reduced or deleted p53 (H520, H1299), wild-type KRAS (H1299), mutated KRAS (A549, H460), wild-type EGFR (A549), mutated EGFR (H1993, H460), and amplified c-met (H1993).. Alpha-tubulin antibodies were purchased from Calbiochem (La Jolla, CA) The secondary antibodies, HRP-conjugated goat anti-rabbit and HRP-conjugated goat anti-mouse, were purchased from Jackson ImmunoResearch (West Grove, PA).. Cell lines All NSCLC (NCI-H520, A549, NCI-H1993, NCI-H460, and NCI-H1299) and SCLC (SHP77 and DMS114) cell lines were obtained from the American Tissue and Cell Collection (ATCC).. 3 mM CDDP in saline (Teva Pharmaceuticals, Israel) was stored at −20°C Antibodies against poly ADP ribose polymerase (PARP), cleaved caspase-3, p-glycoprotein (Pgp; MDR1), and B-cell lymphoma 2 (Bcl-2) were purchased from Cell Signaling Technology (Beverly, MA).. Reagents and antibodies CFZ, provided by Onyx Pharmaceuticals, Inc , an Amgen subsidiary (South San Francisco, CA), was dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. 518b7cbc7d
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