Supplementary Figure S13 from Autocrine 17-β-Estradiol/Estrogen Receptor-α Loop Determines the Response to Immune Checkpoint Inhibitors in Non–Small Cell Lung Cancer

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Switch-off/on EGFR activity in NCI-H1385 cells. a. Experimental scheme: Low EGFR activitywild-type NCI-H1385 cells were knocked-out (KO) for endogenous EGFR by a CRISPR/Cas vector, then stably transfected with an expression vector encoding L858R EGFR. b. Immunoblot of EGFR in wild-type (wt) NCI-H1385 cells and in clones subjected to two (#1, #2) EGFR-CRISPR/Cas vectors. Tubulin is included as control of equal protein loading. The image is representative of one of three experiments. c. L858R EGFR mRNA levels in wt NCI-H1385 cells and in cells stably transfected with the L858R EGFR-expression vector, measured by RT-PCR (technical triplicates). Data are means + SD (n=3, biological replicates). ***p<0.001: L858R cells versus wt cells (ANOVA). d. Immunoblot of EGFR in wt and L858R NCI-H1385 cells. Tubulin is included as control of equal protein loading. The image is representative of one of three experiments. e. EGFR kinase activity, measured in wt and L858R NCI-H1385 cells by a chemiluminescence-based assay in technical duplicates. Data are means+SD (n=3, biological replicates). ***p<0.001: L858R cells versus wt cells (ANOVA).

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