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3 protocols using trcn0000003810

1

H. pylori Infection Model in MKN28 Cells

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MKN28 human gastric epithelial cell line (JCRB Cell Bank, JCRB0253) was a kind gift from Cyril Benes. MKN28 cells were cultured in growth medium, RPMI-1640 medium containing 10% FBS (Invitrogen) and 100 U/mL penicillin and 100 μg/mL streptomycin, at 37°C with 100% humidity and 5% CO2. H. pylori strains NCTC 11637 (ATCC 43504), 26695 (ATCC 700392), J99 (ATCC 700824), BCM-300 (ATCC BAA-1606) and BCS 100 (ATCC BAA-945) were obtained from ATCC and were grown in Brucella broth with 5% FBS at 37°C. For H. pylori infection assays, bacteria cells were harvested by centrifugation at 8000 rpm for 5 minutes and added to gastric cells at multiplicity of infection (MOI) of bacteria to cell from 25:1 to 500:1. Lonp1 polyclonal antibody (HPA002192), β-actin monoclonal antibody (A5441), and pLKO.1-puromysin shRNA lentiviral vectors for Lonp1 (TRCN0000291803) and HIF-1α (TRCN0000003810) were purchased from Sigma. ATP5A monoclonal antibody used for tracking mitochondria (ab14748) was obtained from Abcam. HIF-1α antibody was purchased from Novus Biologicals (NB100-105). Lentiviral overexpression vector pLJM1-EGFP (19319) and pcDNA3 vector containing full-length HIF-1α (18949) were obtained from Addgene. pCMV6-XL4 vector containing full-length Lonp1 (SC117111) was purchased from Origene.
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2

Silencing HIF-1α and HIF-2α in Cancer Cells

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HIFs were silenced with pLKO.1 lentiviral shRNA constructs (HIF-1α: TRCN0000003808, TRCN0000003809, TRCN0000003810; HIF2α: TRCN0000003803, TRCN0000003807, TRCN0000003804) obtained from Sigma, Inc. To produce viral supernatants, we transfected subconfluent monolayers of HEK293T cells with pLKO.1 sh-RNA, pMD2.G, and pCMV-dR8.74 (Shankar et al., 2016 (link)). Viral supernatants were collected between 24 and 72 ​h after transfection. PC-3 and 786-O cells were transduced overnight with viral supernatant (MOI ​= ​0.5) in the presence of 8 ​μg/ml polybrene for 24 ​h, and 24 ​h after replacing with fresh growth medium, cells were selected by treatment with 1.5 ​μg/ml puromycin for four days or until 100% death of the non-transduced cells.
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3

Lentiviral Knockdown of HIF1α and NRF2 in SUM149 Cells

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Lentiviral mission shRNA clones against HIF1α (TRCN0000003808, TRCN0000003809, TRCN0000003810, TRCN0000003811 and TRCN0000010819), NRF2 (TRCN0000007555 and TRCN0000007557) were purchased from Sigma. Recombinant lentiviruses expressing HIF1α or NRF2 shRNA as well as scrambled sequence were packaged at the University of Michigan Vector Core. Parental SUM149 BC cells were infected with recombinant lentiviruses at the presence of polybrene (8μg/ml, Millipore) overnight and the medium containing viruses was changed and replaced with fresh medium after 20h of infection. Puromycin (Invitrogen) selection was performed using 2.5μg/ml for 1 week to select lentiviral vector transduced cells.
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