Fig 1: ESCRT-III-silenced cDCs display increased antigen export to the cytosol(A) Timeline for the ß-lactamase assay in (B) and (C).(B) Representative plots after 3 h of incubation with or without ß-lactamase (gated on live cells).(C) Quantification of 4 independent experiments (two-way ANOVA, mean ± SEM, exact p values are indicated). The zero time point corresponds to incubation for 5 h without ß-lactamase at 37°C.(D–F) ESCRT-III-deficient or control MutuDCs were pulsed with tetramethylrhodamine-labeled dextrans (3K [D], 10K [E], or 70K [F]) before extensive washes and subsequent chase. Representative images acquired with a confocal microscope equipped with a 40× objective are shown (scale bar, 5 µm). Quantification of the median of cytosolic fluorescence and of the ratio of dextran+ vesicle area on total cell area are shown (Student’s t test, mean, exact p values are indicated). Each dot represents one cell. One experiment representative of 2 independent experiments is shown. Arrowheads indicate cells showing cytosolic dextran fluorescence. Because the images shown in (D) originate from the same experiment as the one presented in Figure S3F, the control shRNA image and values are identical (and copied) in both figures.(G) qRT-PCR expression analysis of endogenous Chmp4b (left panel) and shRNA-resistant Chmp4b (right panel) in MutuDCs expressing (resistant CHMP4b) or not expressing (empty vector) shRNA-resistant CHMP4b and the different shRNA. n = 3 (unpaired Student’s t test, exact p values are indicated).(H) Representative flow cytometry plots after 3 h of incubation with ß-lactamase (gated on live cells).(I) Quantification of 2 independent experiments (two-way ANOVA, mean ± SEM, exact p values are indicated).(J) Representative galectin-3 staining images obtained with a confocal microscope equipped with a 40× objective (scale bar, 5 µm). n = 2. Arrowheads indicate locations of galectin-3 foci.