Sample 35
Mus musculus
Protocols: This submission studied the inhibition of PARP14 under IFNγ stimulation in three cell lines. A375 (https://www.cellosaurus.org/CVCL_0132) human melanoma cell line was maintained in DMEM (high glucose) medium. MC38 (https://www.cellosaurus.org/CVCL_B288) mouse colon cancer cell line was maintained in RPMI-1640 (Sigma-Aldrich) medium. YUMM2.1 (https://www.cellosaurus.org/CVCL_JK30) mouse melanoma cell line was maintained in RPMI-1640 (Sigma-Aldrich) medium. All growth media were supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% penicillin-streptomycin (P/S; Sigma-Aldrich) and 0.1% by volume of DMSO (solvent for the inhibitors). All cells were maintained under standard conditions at 37°C in a 5% CO2 humidified incubator and passaged before reaching confluency. Cell cultures were routinely tested for mycoplasma contamination by PCR. For chronic IFNγ stimulation, human cells (A375) were treated with 20 IU/ml, mouse cells (YUMM2.1 and MC38) with 50 IU/ml, replacing medium and IFNγ every 72 h with passaging of cells as necessary for 2 weeks. PARP14 inhibition was with 100 nM RBN012759 or 500 nM RBN012811 for the last 48 h. Recombinant human IFNγ (PHC4031) and recombinant mouse IFNγ (PMC4031) were purchased from Gibco and used at the indicated concentrations. The PARP14 inhibitors RBN012759 and RBN012811 were kindly provided by Ribon Therapeutics and used at the indicated concentrations. This submission studied the inhibition of PARP14 under IFNγ stimulation in three cell lines. A375 (https://www.cellosaurus.org/CVCL_0132) human melanoma cell line was maintained in DMEM (high glucose) medium. MC38 (https://www.cellosaurus.org/CVCL_B288) mouse colon cancer cell line was maintained in RPMI-1640 (Sigma-Aldrich) medium. YUMM2.1 (https://www.cellosaurus.org/CVCL_JK30) mouse melanoma cell line was maintained in RPMI-1640 (Sigma-Aldrich) medium. All growth media were supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% penicillin-streptomycin (P/S; Sigma-Aldrich) and 0.1% by volume of DMSO (solvent for the inhibitors). All cells were maintained under standard conditions at 37°C in a 5% CO2 humidified incubator and passaged before reaching confluency. Cell cultures were routinely tested for mycoplasma contamination by PCR. For chronic IFNγ stimulation, human cells (A375) were treated with 20 IU/ml, mouse cells (YUMM2.1 and MC38) with 50 IU/ml, replacing medium and IFNγ every 72 h with passaging of cells as necessary for 2 weeks. PARP14 inhibition was with 100 nM RBN012759 or 500 nM RBN012811 for the last 48 h. Recombinant human IFNγ (PHC4031) and recombinant mouse IFNγ (PMC4031) were purchased from Gibco and used at the indicated concentrations. The PARP14 inhibitors RBN012759 and RBN012811 were kindly provided by Ribon Therapeutics and used at the indicated concentrations. Total RNA was isolated using the RNAeasy mini kit. RNA integrity was assessed on the Agilent 2200 TapeStation (Agilent Technologies). TruSeq Stranded mRNA assay (Illumina)