Journal: Communications Biology
Article Title: Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression
doi: 10.1038/s42003-025-08983-z
Figure Lengend Snippet: a Western blots showing nuclear NRF2 protein levels in cells expressing control and KEAP1-targeting sgRNAs. b , c Flow-cytometry data (quantified in bar plots as median fluorescence intensity) shows cell surface PD-L1 expression following NRF2 activator DMF treatment in IFNγ-induced MiaPaca2 and ASPC1 cells (100 µM, 48 h), (n = 3 replicates, unpaired t-test). d Western blot showing NRF2 protein levels in response to increasing doxycycline concentrations that drive NRF2 over-expression in MP2 cells. e Flow-cytometry data (quantified in bar plots as median fluorescence intensity) shows surface PD-L1 expression in response to increasing doxycycline concentrations that drive NRF2 over-expression (n = 3 replicates, unpaired t-test). f Western blots showing NRF2 and NQO1 (known NRF2 target) protein levels in cells expressing control and NRF2 targeting sgRNAs in MP2 cells. g The bar plot shows qRT-PCR measured PD-L1 mRNA levels in cells expressing control and NRF2-targeting sgRNAs (n = 3 independent experiments, unpaired t-test). h Flow-cytometry data (quantified in bar plots as median fluorescence intensity) shows surface PD-L1 expression of control and NRF2 knock-out cells with and without Bardoxolone treatment in the presence of IFNγ in MP2 cells (n = 3 replicates, unpaired t-test). i The bar plot shows chromatin immunoprecipitation followed by qPCR fold enrichment levels of NRF2 at the PD-L1 enhancer element control and KEAP1 knock-out MP2 cells (n = 3 independent experiments, unpaired t-test).
Article Snippet: Cells were treated with 50 ng/mL recombinant IFNγ (human: PeproTech, #300-02 and mouse: PeproTech, #315-05) for 48 h.CDK1 inhibitor RO-3306 (Selleck Chemicals, #S7747) treatment was 5 μM for 24 h and CSNK1A1 inhibitor D4476 (Millipore, #218696) treatment was 2.5 μM for 48 h. Cells were treated with 2.2 μM Bardoxolone (MedChem Express, #HY-14909) or CDDO-me (Tocris, #6646) for 72 h. DMF (MedChem Express, #CS-0909) treatment was 100 μM for 48 h. ATRA (StemCell Technologies, #72262) treatment was 1 μM for 48 h. Cells were treated with indicated doses of Doxycycline (Sigma, #D9891) for 48 h to drive NRF2 over-expression.
Techniques: Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Over Expression, Quantitative RT-PCR, Knock-Out, Chromatin Immunoprecipitation
