Lookout mycoplasma pcr kit

HEK293T human embryonic kidney cells, HCT116 human colon carcinoma cells (containing wild-type p53: p53^wt/wt HCT116, further noted as HCT116; lacking p53: p53^−/− HCT116 and p53^R248/− HCT116, a kind gift from Prof. Karen Vousden, Glasgow), and the human colon cancer cell line LIM1215 (a kind gift from Prof. Sabine Tejpar, Leuven) were maintained at 37°C at 95% air and 5% CO₂ in DMEM (Gibco, life technologies) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM l-glutamine (Gibco), and 1% penicillin/streptomycin (PenStrep, Gibco). All cancer cell lines underwent mycoplasma testing before their use. Negative mycoplasma contamination status was verified using LookOut Mycoplasma PCR Kit (Sigma) and MycoAlert Mycoplasma Detection Kit plus Assay Control (Lonza). All cell lines were not maintained longer than 10 passages in culture to perform experimental procedures. Transfection of siRNA was performed with lipofectamine RNAiMAX (Life Technologies) according to the supplier’s protocol. Catalog numbers for the different siRNAs used can be found in Table 1.

The REDD1^−/− allele was generated as previously described (Sofer et al. 2005 (link)). MEFs not harboring LSL-Kras^G12D were immortalized by retroviral transduction of SV40 large T-antigen unless indicated otherwise, and were maintained in DMEM/10% FCS/Pen/Strep. Human non-small cell lung cancer cell line A549 was obtained from the American Type Culture Collection and maintained in DMEM/10% FCS/pen/strep. Primary pancreatic intraepithelial cells (AH375) were isolated from LSL-KRAS^G12D mice as described previously (Corcoran et al. 2011 (link)). All pancreatic ductal epithelial cells were routinely maintained in ductal media on laminin-coated plates. Negative mycoplasma contamination status of all cancer cell lines, 293T cell line and primary cells used in the study was established using LookOut mycoplasma PCR kit (Sigma MP0035).

The human glioma cells line viz, LN-18, U87MG, SNB-19, and U-251 were procured from ATCC and cultured in ATCC-recommended complete media. Whereas human neural glial cell-line (HNGC-2) and primary culture derived from GBM tumors (G1) were generated at National Centre for Cell Science (NCCS), Pune. Experimental methods on established patient-derived xenograft (PDX) cells CNXF-2599L were conducted at Charles River, Germany. The cells were maintained in a humidified chamber at 37 °C and 5% CO₂. All the compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM and diluted in a culture medium immediately before use. Pyronaridine tetraphosphate (#VIA-404019) was procured from Chemical Centre (Mumbai, India) while Doxorubicin and Ritonavir were available in-house. Dulbecco’s phosphate-buffered saline without calcium and magnesium (#14025092), cell culture media, and FBS (#26140079) were purchased from Gibco (Thermofisher, Waltham, MA, USA). Lookout mycoplasma PCR kit (#MP0035) and PMS (#P9625) were from Sigma while 96-well flat-bottomed white polystyrene plates were from Corning (#3912) and MTS reagent from Promega (Madison, WI, USA, #G111).