INCA033989 antagonistic effect is dependent on cell internalization. (A) Time-dependent binding of Alexa Fluor 647–INCA033989 to Ba/F3-TPOR/CALR^del52 cells, measured using high-content imaging. Representative images are shown at a 20× magnification. Scale bar = 10 μm. (B-D) Ba/F3-TPOR/CALR^del52 cells were preincubated for 10 minutes with dynasore or dimethyl sulfoxide (DMSO), followed by treatment with Fabfluor-labeled INCA033989 or -isotype for 24 hours (see supplemental Methods for further details). (B) Internalization rate of Fabfluor-pH sensitive–labeled antibodies over time. (C) Cell proliferation over time, calculated as the cell confluence. (D) Caspase induction over time. (E) Ba/F3-TPOR/CALR^del52 cells were treated with INCA033989 premixed with dynasore (inhibitor of dynamin-dependent endocytosis) or DMSO and incubated for 3 hours and levels of pSTAT5 were assessed by Meso Scale Discovery. Inhibition of pSTAT5 was calculated after normalization to the maximal (100%) inhibition achieved by the cell treatment with 6 μM of ruxolitinib and no inhibition (0%) in DMSO-treated cells. The data are representative of 3 independent experiments. (F) INCA033989 is internalized upon binding to mutCALR on the cell surface. Ba/F3-TPOR/CALR^del52 cells were treated with Alexa Fluor 647–INCA033989 for 18 hours and the microscopy images were evaluated. Green: cell membrane; blue: nucleus; magenta: INCA033989. (G) INCA033989 colocalizes with lysosomal markers upon internalization. Ba/F3-TPOR/CALR^del52 cells were pretreated with Alexa Fluor 647–INCA033989 for 2 hours on ice, followed by washing and additional incubation for 6 hours at 37°C. The lysosomal compartment was stained with anti-LAMP-2 and the cells were evaluated by microscopy. Green: LAMP-2; blue: nucleus; magenta: INCA033989; yellow: colocalization of LAMP-2 and INCA033989. LAMP-2, lysosome-associated membrane protein 2.