Fig. 6: Zeb1 controls phagosomal ROS-dependent rupture of phagosomal membrane in cDC1.

a WT or Zeb1-deficient Flt3L-cDC1, or Zeb1-deficient Flt3L-cDC1 lentivirally transduced with Cybb containing synonymously mutated miR-96 binding sites were cultured for 3 days with CFSE-labeled OT-I T cells and different dose of HKLM-OVA and assayed for OT-I proliferation and activation (CFSE^-CD44⁺). b WT Flt3L-cDC1 retrovirally transduced with empty vector (EV) or miR-96 or miR-182 were cultured and analyzed as described in (a), with various dose of HKLM-OVA as antigens. c Intracellular and mitochondrial ROS production in WT and Zeb1-deficient Flt3L-cDC1 challenged with HKLM-OVA for indicated times, were measured by flow cytometric analysis of CellROX and MitoSOX fluorescence. The mean fluorescent intensity (MFI) of CellROX and MitoSOX fluorescence were quantified at the bottom. d, e Phagosomal ROS production in WT and Zeb1-deficient Flt3L-cDC1 exposed with OxyBURST/Alexa Fluor 647-conjugated HKLM-OVA for indicated times, were measured by flow cytometric analysis (d) of OxyBURST and Alexa Fluor 647 fluorescence. The phagosomal ROS production was quantified as the ratio of OxyBURST⁺ cells to Alexa Fluor 647⁺ cells (e, top row) or MFI of OxyBURST in Alexa Fluor 647⁺ cells (e, bottom row). f WT and Zeb1-deficient Flt3L-cDC1 were cultured with HKLM-OVA for 30 min, and treated with or without DPI (10 μM) for 4 h before addition of CFSE-labeled OT-I T cells, and assayed for OT-I proliferation and activation (CFSE^-CD44⁺). g, h Confocal microscope images of mCherry::galectin-3-expressed WT and Zeb1-deficient Flt3L-cDC1 challenged with Alexa Fluor 647-labeled HKLM-OVA. Colocalizing signal of Galectin-3⁺HKLM⁺ cells were counted and plotted as a ratio of total HKLM⁺ cells. Each symbol represents an individual sample, small horizontal lines indicate the mean (±s.d.). Data are representative of three independent experiments. Data are presented as mean ± s.d. Statistical analysis was performed using Two-tailed unpaired Student’s test (h) or two-way ANOVA with Tukey’s (a, f) or Sidak’s (b, e) multiple comparisons test. Source data are provided as a Source Data file.