The following tumour cell lines were purchased from ECACC directly from Sigma-Aldrich:breast: MDA-MB-231[16 (link)](92020424), MCF7[17 (link)] (86012803); colon: HT29[18 (link)] (91072201), SW480[18 (link)] (87092801), SW620[18 (link)] (87051203); lung: A549[19 (link)] (86012804) and prostate: PC-3[20 (link)] (90112714); as well as endothelial cells: HUVEC (S200-05N) and leucocytes: Jurkat cells[21 (link)] (88042803).The brest cancer tumour cell HCC1937 lines[22 (link)](ATCC-CCL-247)and HCC1954[23 (link)] (ATCC CRL-2338) were purchased from ATCC; the endometrial cell lines HEC1A and HEC1A stably expressing the ETV5 transcription factor (HEC1A-ETV5) were previously described [24 (link)–26 (link)]. Medium for cell culture were obtained from Gibco (DMEM: SW480, SW620, A549, MCF7, MDA-MB-231, PC-3; McCoyy᾿s: HT29, HEC1A, HEC1A-ETV5; RPMI 1640: HCC1937, HCC1954,Jurkat), and from Lonza (Basel, Switzerland) (EGM-2: Huvec). The medium was supplemented with 10% FBS and 1% streptavidin-penicillin (Life Technologies, Carlsbad Ca, USA). Cells were maintained at 37°C in a 5% CO2 incubator. For selected experiments, we transduced MDA-MB-231 and SW480 cells with lentiviral transduction particles (turboGFP, Sigma-Aldrich) to establish cells that expressed green fluorescent protein (GFP); cells were further selected with puromycin (5 μg/mL) and checked by flow cytometry (96%).
Turbogfp
Manufactured by Merck Group
TurboGFP is a variant of the green fluorescent protein (GFP) that exhibits enhanced fluorescence intensity and stability. It is a useful tool for a variety of research applications, such as protein localization studies, cell lineage tracing, and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Mcf 7, by Merck Group (1 mentions)
Mda mb 231, by Merck Group (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Sw620, by American Type Culture Collection (1 mentions)
Hcc1954, by American Type Culture Collection (1 mentions)
Sw480, by Thermo Fisher Scientific (1 mentions)
Hec1a, by Lonza (1 mentions)
Hcc1937, by Lonza (1 mentions)
Hcc1954, by Lonza (1 mentions)
Penicillin streptavidin, by Thermo Fisher Scientific (1 mentions)
Ht 29, by American Type Culture Collection (1 mentions)
Mda mb 231, by Lonza (1 mentions)
Ht 29, by Lonza (1 mentions)
Sw620, by Lonza (1 mentions)
Pcbascei, by Addgene (1 mentions)
Pdrgfp, by Addgene (1 mentions)
X tremegene 9 reagent, by Roche (1 mentions)
Magmax 96 total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using turbogfp
1
Establishing Diverse Cell Line Panel
2
Evaluating HR and NHEJ in Rif1-/- Cells
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For determining the HR and NHEJ proficiency in WT and Rif1−/− cells the following plasmids were used: pDRGFP (Addgene plasmid #26475) and pCBASceI(Addgene plasmid #26477) for HR and pCVL Traffic Light Reporter1.1(Sce target) (Addgene plasmid #31482) along with pCBASceI for NHEJ. For measuring and controlling transfection efficiency, Turbo-GFP expressing plasmid (Sigma, MISSION SHC003) and Scrambled plasmid (Sigma, MISSION SHC002), were used. In all, 2.5 x 105 cells were co-transfected with 3 μg of plasmid combinations (i.e., 1.5 μg of each plasmid) using Xtremegene-9 reagent from Roche in six-well dish. Cells were transfected twice at an interval of 24 h. Post 48 h of transfection, cells were harvested and the GFP-positive cells (for HR) and RFP-positive cells (for NHEJ) were assessed by flow cytometry. Fifty thousand events were recorded for each sample. Background normalization was done by using samples co-transfected with scrambled—reporter plasmid and also scrambled—pCBASceI plasmid. Final percentage of GFP and RFP-positive cells were calculated based on the transfection efficiency of the cells. Each experiment was independently performed at least thrice.
3
Production and Purification of Lentiviral Constructs
4
Targeted shRNA Knockdown in Cell Lines
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For the shRNA studies, cell suspensions at 500 cells per well were first dispensed into 384-well microtiter plates in 45 µL media. After overnight incubation, media was aspirated from the assay plates and replaced with media plus 8 µg/mL of polybrene. TRCN#40273 targeting CTTN (Sigma-Aldrich) was thawed from storage at room temperature and diluted in plain DMEM; after which 1 µL was transferred into the assay plates to achieve a final MOI of 4. The assay plates were briefly centrifuged for 8 min at a speed of 1,300 rpm. At day 3 post-transduction, media was aspirated and 45 µL of media containing puromycin was added to the cells and further incubated to complete selection. At day 7 post-selection, cells were harvested using the MagMAX-96 Total RNA Isolation Kit (Thermo Fisher Scientific) following manufacturer’s specifications. As internal reference, we included non-transduced cells with no puromycin, Non-Target shRNA #1 (Sigma-Aldrich), and TurboGFP (Sigma-Aldrich) as controls for transduction assessment.
5
Generation of Repair-Deficient Cell Lines
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Stable cell lines defective in the different repair pathways (see Table 1 for details) were generated in HCT116 p53−/− cells24 (link) according to standard protocols using Mission short hairpin RNA (shRNA) lentiviral particles bearing the pLKO.1-puro vector either containing the turboGFP, TagCFP or TagRFP gene (Sigma-Aldrich). Targeted genes and shRNA sequences are listed in Table 1 . Briefly, HCT116 cells were infected with lentiviral particles for 24 h, then puromycin was applied to select for lentiviral transduction. Puromycin resistant cells were diluted (limit-dilution), and fluorescent clones were selected, amplified and screened for the shRNA-mediated depletion efficiency by Western blot and RT-qPCR analysis.
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