Rpmi media

BM-derived DCs (BMDCs) were prepared as follows. BM cells were flushed from femur and tibia of WT C57BL/6J mice and cultured in RPMI media (Sigma) containing 10% FSC and 1% Penicillin/Streptomycin (Gibco) supplemented with GM-CSF (5 ng/ml). Fresh media containing GM-CSF was added at day 3 and 5 of cultivation. After 7 days, BMDCs was pulsed with OVA cognate peptide 323-339 (irrelevant OVA 257–264 peptide was used as control) (InvivoGen) at a concentration of 1 μg/ml and co-cultivated with OVA-specific OT-II T cells and Tregs (10 000 BMDCs: 50 000 OT-II T cells: 50 000 Tregs). OT-II T cells were isolated from OT-II⁺Rag1^−/− mice as MACS-enriched CD4⁺ T cells (CD4⁺ T Cell Isolation Kit, Miltenyi biotec). CD4⁺ conventional T cells (Tconv) were used as a negative control. Tregs were isolated from LNs (pLN and mLN) of WT (MyD88^fl/fl) and MyD88^ΔTECs mice using subsequent Auto-MACS (Miltenyi biotec) procedure. CD4-enriched T cells (CD4⁺ T Cell Isolation Kit, Miltenyi biotec) were stained by anti-CD25 biotin conjugated antibody and CD4⁺CD25⁺ Tregs were isolated using Anti-Biotin MicroBeads (Miltenyi biotec). Tconv cells were prepared using Auto-MACS as CD4⁺CD25^– cells. After 3 days of co-cultivation, cells were stained with anti-Vβ5 and anti-Vα2 antibodies to distinguish OT-II⁺ T cells. Proliferation was measured by FACS using CPD670 staining.

Initially, cells were seeded at a density of 5 × 10³ cells/96 well. After 24 h of incubation, the proliferation ability of PDAC cells under different treatments was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). BxPC3 or PANC-1 cells were initially silenced or overexpressed for either hnRNPC or IQGAP3. Further, the cell growth assay was performed at 1, 2, 3, 4, 5, and 6^th day. Each day, the cells were incubated with 10 μL MTT (5 mg/mL) in RPMI media (sigma Aldrich, St. Louis, Missouri, USA) for 4 hours. Further, the crystals in the well were dissolved by 100 μL dimethyl sulfoxide (DMSO) for 10 mins (Sigma Aldrich, St. Louis, Mo., USA). Subsequently, the optical density (OD) was measured at 490 nm on the Microplate Reader (Bio-Rad, Hercules, CA, USA).

Human NSCLC lines A549, H460, H1944, H1792, H23, H1299, H1975 and H1650 were purchased from LGC Ltd. H460 FLuc were purchased from PerkinElmer. H1299 FLuc were purchased from AMS biotec. A549 NRF2 knockout (KO), A549 KO-restored (KO-R), H1299 T80K and H1299 PLX317 were previously described ^{24 , 25 (link)}. To pharmacologically activate NRF2 expression, cells were treated with 100 nM of the KEAP1 inhibitor, KI696, for 24 h. All cells were cultured in RPMI media (Sigma-Aldrich), supplemented with 10% foetal bovine serum (ThermoFisher Scientific) and 100 U.mL⁻¹ penicillin and 100 μg.mL⁻¹ streptomycin (Sigma-Aldrich), maintained at 37 °C and 5% CO₂. Cell lines were tested monthly for mycoplasma infection (Eurofins).
Cells were seeded for 24 h prior to experimental endpoint in 6-well plates in 2 mL of media. All cells were seeded at 1.5 × 10⁵/mL, except for H1944 and H1792, which were seeded at 2.75 × 10⁵/mL; H1650, which was seeded at 1.75 × 10⁵/mL; and H23 and H1975, which were seeded at 2.5 × 10⁵/mL. For KI696 treatments, H1650, H23, H1975 and H1299 were seeded at 1.0 × 10⁵/mL, 1.0 × 10⁵/mL, 1.25 × 10⁵/mL and 0.75 × 10⁵/mL, respectively 2 mL of media 24 h prior to treatment.

Human embryonic stem cell (ESC) lines were obtained with consent either directly from the derivation laboratory or the UK Stem Cell Bank. Cells were maintained on inactivated mouse embryonic fibroblast (MEF) cells [21] . The differentiation protocol (Fig. 1) was commenced 3–4 days post passage onto fresh MEFs using Wnt3a (R&D Systems, UK) and Activin A (Peprotech, UK), diluted in RPMI media (Sigma-Aldrich, UK); followed by BMP2, OSM, FGF2, HGF (all R&D Systems) and DEX (Sigma-Aldrich, UK), diluted in Hepatocyte Culture Medium (HCM) (Lonza, UK). Information on the human fetal and adult hepatocyte controls can be found in the Supplementary Materials and methods. Human induced pluripotent stem cells (IPSCs) were developed and differentiated as previously reported [6] (link), [22] (link).Fig. 1

The three-stage differentiation protocol. RPMI, Roswell Park Memorial Institute; FBS, fetal bovine serum.