Pancreatic NET cell lines QGP-1 and BON-1 were purchased from the American Type Culture Collection (Manassas, Virginia, USA). SKBR3 and MBA-MD-231 were used as control cell lines (Supplementary Materials). Cells were incubated in hypoxic conditions using the Whitley H35 hypoxystation (Don Whitley, Shipley, West Yorkshire, UK) in standard media in an atmosphere of 1% O2, 94% N2 and 5% CO2 for 24 hours and lysed in parallel with normoxic controls. Protein expression was evaluated by immunoblotting (Supplementary Methods).
Bon 1
Manufactured by American Type Culture Collection
Sourced in United States
The BON-1 is a bioreactor system designed for the cultivation and propagation of various cell lines and microbial cultures. It provides a controlled environment for the growth and maintenance of these biological samples. The BON-1 facilitates the regulation of key parameters such as temperature, pH, and dissolved oxygen levels to support the optimal growth conditions for the cultured organisms.
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3 protocols using bon 1
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Hypoxic Regulation of Pancreatic NET Cells
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Metastatic Pancreatic NET Cell Line BON1
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The human metastatic, adherent pNEN cell line, BON1, was obtained from the American Type Culture Collection and grown as a monolayer in 75 cm2 flasks (Corning, NY, USA) in a 1:1 mixture of RPMI 1640 and Ham’s F-12 media supplemented with 10% foetal bovine serum, penicillin and streptomycin (100 IU/mL) at 37°C with 5% CO2 (30 (link)). To investigate the role of interleukin-6 (IL-6) in the activation of STAT3, BON1 cells were treated with recombinant human IL-6 (25 ng mL−1) (Cell Signaling Technology) for different periods (24 h, 48 h, 72 h). The period (72 h) at which IL-6 exerted its maximum effect on BON1 cells was chosen for study.
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Modulation of PCSK9 expression in p-NENs cell lines
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Human p-NENs cell lines BON-1 was obtained from American Type Culture Collection and authenticated with short tandem repeat (STR, JiaPeng Co., Ltd, China). Cells were cultured in DMEM medium (Hyclone) added with 10% FBS (Siencell, USA) conventionally.
The miRNA-224 agomir, agomir negative control, PCSK9 siRNA (siPCSK9) and siRNA negative control (siNC) were designed (GenePharma Co.,Ltd, China). The sequence of miRNA-224 agomir was 5′-CAAGUCACUAGUGGUUCCGUU-3′ (sense), 5′-CGGA ACCACUAGUGACUUGUU-3′ (antisense). Sequence of siRNA against PCSK9 was shown as follows: 5′-CCAAGAUCCUGCAUGUCUUTT-3′ (sense), 5′-AAG ACAUGCAGGAUCUUGGTT-3′ (antisense). Sequence of siRNA negative control was as follows: 5′-UUCU CCGAACGUGUCACGUTT-3′ (sense), 5′- ACGU GACACGUUCGGAGAATT-3′ (antisense). Transfected experiments were performed with Lipofectamine 2000 (Invitrogen) according to manufacturer's procedure. BON-1 cells were respectively cultured with miRNA agomir, siRNA, or negative control for 48 hours.
The miRNA-224 agomir, agomir negative control, PCSK9 siRNA (siPCSK9) and siRNA negative control (siNC) were designed (GenePharma Co.,Ltd, China). The sequence of miRNA-224 agomir was 5′-CAAGUCACUAGUGGUUCCGUU-3′ (sense), 5′-CGGA ACCACUAGUGACUUGUU-3′ (antisense). Sequence of siRNA against PCSK9 was shown as follows: 5′-CCAAGAUCCUGCAUGUCUUTT-3′ (sense), 5′-AAG ACAUGCAGGAUCUUGGTT-3′ (antisense). Sequence of siRNA negative control was as follows: 5′-UUCU CCGAACGUGUCACGUTT-3′ (sense), 5′- ACGU GACACGUUCGGAGAATT-3′ (antisense). Transfected experiments were performed with Lipofectamine 2000 (Invitrogen) according to manufacturer's procedure. BON-1 cells were respectively cultured with miRNA agomir, siRNA, or negative control for 48 hours.
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