Fig. 5: EGFR trafficking upon CYpHER treatment.
From: CYpHER: catalytic extracellular targeted protein degradation with high potency and durable effect
a Designs of various EGFR CYpHERs and control molecules. b A549-EGFR-GFP (knockin) cells treated with PBS or 10 nM CYpHER for 48 h with drug and imaged for GFP localization. c A549 cells treated with 2 or 10 nM CYpHER for 24 hrs, or 10 nM CYpHER for 24 h followed by 24 h drug withdrawal, and assayed by flow cytometry for surface EGFR per cell. N cells per sample as follows: Untreated, 3598. CT-1212-1: 2 nM, 2979; 10 nM, 3288; Withdrawal [WD], 2795. CT-1112-1: 2 nM, 3527; 10 nM, 3286; WD, 3135. CT-1211-1: 2 nM, 2339; 10 nM, 3018; WD, 2250. CT-1111-1: 2 nM, 3412; 10 nM, 3532; WD, 2516. All-vs-all within treatments (including Untreated) by Kruskal–Wallis [KW] test: CT-1212-1, 2 nM vs 10 nM P = 0.1494, all others P < 0.0001; CT-1112-1, 2 vs 10 nM P = 0.0031, all others P < 0.0001; CT-1211-1; 2 vs 10 nM P = 0.0002, all others P < 0.0001; CT-1111-1, all P < 0.0001. d A549-EGFR-GFP cells imaged for GFP localization after 24 h treatment with 10 nM CYpHER (CT-1212-1) or control molecule (CT-1232-1 or CT-3212-1). e A549-EGFR-GFP cells imaged for GFP localization after 20 min treatment with PBS or 10 nM CT-1212-1. f A549-EGFR-GFP cells treated without or with 10 μM human holoTF for 15 min followed by addition of PBS or 10 nM CT-1212-1 for 4 h and imaged for GFP localization. g Same experimental design as in f except altered amount of holoTF and analyzed by flow cytometry for surface EGFR. Dashed lines indicate quantitation of surface EGFR in untreated cells (upper) or cells treated with 10 nM CT-1212-1 but no holoTF (lower). Each treatment vs No CYpHER (100% line) by Kruskal–Wallis test with Dunn’s correction [KWD] was P < 0.0001. Each CYpHER + holoTF treatment vs CYpHER + no holoTF (“No holoTF” line at ~21%) by KWD: 100 nM holoTF P = 0.9831, all others P < 0.0001. Experiments in b–g were performed once (c, f, g) or twice (b, d, e) with similar results. All box plots (c) feature a median (black line), 25th and 75th percentiles (box boundaries), and 5th and 95th percentiles (whiskers). See the Supplementary Data for full statistical breakdown. Source data are provided as a Source Data file.
