Journal: Nature Communications

Article Title: The transcription factor Zeb1 controls homeostasis and function of type 1 conventional dendritic cells

doi: 10.1038/s41467-023-42428-7

Figure Lengend Snippet: 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.

Article Snippet: The expression plasmid for mCherry:Galectin-3 (Cat#85662) was purchased from Addgene.

Techniques: Transduction, Binding Assay, Cell Culture, Labeling, Activation Assay, Plasmid Preparation, Fluorescence, Microscopy, Two Tailed Test