ATAT1 is required for efficient antigen extraction and presentation by B cells. (A) Time-lapse images of control (siCTL) or ATAT1-silenced (siATAT1) B cells expressing cathepsin D-RFP, activated on slides containing BCR⁺ ligand plus DQ-OVA (green) during 30 min. Images show time points from 15 to 30 min. (B and C) siCTL and siATAT1 B cells activated for 30 min on BCR⁺ ligand–DQ-OVA–coated slides. Representative images show MR and DQ-OVA staining at the synaptic interface, with corresponding fluorescence intensity maps. (B) Top graph: Quantification of MR fluorescence distribution in z-scan. (B and C) Radial distribution of fluorescence intensities according to their accumulation from the center to the periphery of the synapse plane. (D–F) Antigen presentation assays of B cells activated on substrates with different stiffness (D) and with control or ATAT1-silenced B cells (F). (E and G) Lack peptide control assays for the conditions shown. Mean amounts of IL-2 are shown for a representative of three independent experiments performed in triplicate. (H) Top: Representative immunofluorescence images of siCTL and siATAT1 cells stained for F-actin (red) and MHC-II (green). Bottom: Quantification of MHC-II at the cell surface by flow cytometry. Left: Representative histogram of MHC-II fluorescence intensity. Right: Quantification of MHC-II surface expression as a percentage relative to siCtl. Scale bars are 10 μm. Data consider n ≥ 30 cells pooled from N = 3 independent experiments. (D–G) Two-way ANOVA with Sidak’s multiple comparison test. (H): t test. P values illustrated with asterisks are * <0.05, ** <0.01, *** <0.001, and **** <0.0001. Error bars are mean ± SEM.