100 mm dishes

The human monocyte/macrophage (THP-1) and human gastric adenocarcinoma cell lines (AGS) were procured from National Centre for Cell Science, Pune, India. AGS and THP-1 cell lines were grown in Ham's-F12 and RPMI 1640 media (Invitrogen Life Technologies), respectively, after supplementing with 10% (vol/vol) heat inactivated fetal bovine serum (FBS) (Invitrogen Life Technologies) and 1% antibiotic–anti-mycotic solution (Invitrogen Life Technologies). Cells were maintained in a humidified incubator with 5% CO₂ at 37°C. THP-1 cells were allowed to differentiate into macrophages with the treatment of 10 ng/ml phorbol myristate acetate (PMA) (Sigma-Aldrich) for 48 h. After 48 h, RPMI 1640 medium was aspirated, replaced with fresh medium with 10% FBS and maintained for another 24 h. For infection experiments, cells were seeded in 6 well plates or in 100 mm dishes (Corning, USA) (70% confluent). Cells were serum starved for 16 h before infection and were later infected with H. pylori at a multiplicity of infection (MOI) of 100.

Lentiviral particles were produced according to previously published protocols (50 (link)). In short, 1 day before transfection, 1.5 × 10⁶ HEK293T cells [American Type Culture Collection (ATCC)] were plated on 100-mm dishes (Corning) in DMEM (PAA Laboratories) supplemented with 10% (v/v) fetal bovine serum (FBS) (PAA Laboratories) and 1× penicillin-streptomycin (PAA Laboratories). The transfection mix consisted of 300 μl of Opti-MEM (Thermo Fisher Scientific), 2.5 μg of transfer vector (pER4; Novartis), 2.5 μg of pCMV-ΔR8.2 (Addgene), 0.3 μg of pCMV-VSV-G (Addgene), and 20 μl of Fugene HD (Promega). Per construct, four dishes were prepared. One day later, the medium was changed. The next day, the supernatant was collected and filtered (0.45-μm pore size; Merck), and the viral particles were enriched using ultracentrifugation (Beckman) at ~140,000g. The pellet was resuspended in 150 μl of Opti-MEM, aliquoted, and stored at −80°C. For each experiment, the needed lentiviral particles were produced in batches to reduce intraexperimental deviations. Each batch was tested for comparable overexpression efficiencies, and the required transduction volumes were determined.

On day 1, HEK293T cells were seeded into 100mm dishes (Corning). On day 2, cells were ∼50%–70% confluent at the time of transfection. For each dish, 6 μg of pHR vector containing the construct of interest, 4 μg of dR8.91 and 1 μg of pMD2.G (Addgene) were mixed in 1 mL of Opti-MEM reduced serum media (GIBCO) with 30 μL of Mirus TransIT-LT1 reagent and incubated at room temperature for 30 minutes. The transfection complex solution was distributed evenly to HEK293T cultures dropwise. On day 5, lentiviruses are harvested from the supernatant with a sterile syringe and filtered through a 0.22-μm polyvinylidene fluoride filter (Millipore).
For A549 cells, lentivirus precipitation solution (Alstem) was added and precipitated as per the manufacturer’s protocol. One well of a 6-well plate (Corning) of A549 cells at ∼50% confluency was transduced with the precipitated lentivirus. After 2-3 days of growth, the cell supernatant containing the virus was removed and the cells were expanded. Cells were then sorted for mCherry+ cells using a Sony SH800S cell sorter.

Barcoded MSH2-2A-bl^R cDNA libraries were packaged into lentivirus by co-transfecting HEK293T/17 cells (ATCC) with the transfer plasmid pool plus envelope and packaging vectors (pMD2.G, Addgene #12259 and psPAX2, #12260; gifts from Dr. Didier Trono). For each pool, 4.4 × 10⁶ cells were plated in a 100 mm dish, then transfected with 17 μg total plasmid (2.0:1.0:1.3 transfer:envelope:packaging ratio), using Lipofectamine 3000 (Thermo). Media was replaced at 6 h post transfection, and viral supernatants were collected at 24 h, passed through an 0.45 μm filter (Millipore), and used immediately or stored at −80°C until use. Viral titer was estimated by transduction with a dilution series followed by puromycin selection and CellTiterGlo (Promega) cell proliferation assay,²⁸ (link) and verified by counting unique barcodes in the transduced population (barc-seq, described below). For each tile, MSH2 knockout HAP1 or HEK293 cells were transduced with mutant library at low multiplicity of infection (<0.1), by applying 1.5 mL viral supernatant (with 8 μg mL⁻¹ polybrene) to each of four 100 mm dishes (Corning) containing ∼7.5 × 10⁶ cells. After 48 h, transduced cells were selected by addition of 1 μg mL⁻¹ puromycin, which was included in all subsequent steps.

Cells were seeded at 1×10⁶ in 100 mm dishes (Corning Incorporated) and cultured for 24 hours. The cells were then treated with either PBS (vehicle control) or PV-10 and cultured for 24 hours, protected from light. Media were collected from cell cultures, and the cells were washed with PBS and collected following trypsinization. The cells were then washed in ice-cold PBS and centrifuged at 1,200 rpm at 4°C for 5 minutes. The supernatant was removed, and the pellet was resuspended in RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate [SDS]) supplemented with 1% (v/v) phosphatase inhibitor (Sigma-Aldrich Co.) and 1% (v/v) protease inhibitor (Sigma-Aldrich Co.). The samples were transferred to 1.5 mL tubes, incubated on ice for 10 minutes, vortexed and centrifuged at 12,000 rpm for 10 minutes. The supernatants were collected as whole cell lysates and either used immediately or stored at −20°C.