cTECs present increased abundance of CLIP-bound I-A ^b complexes in the absence of Vps34. (A–C) cTECs (CD45⁻EpCAM⁺Ly-51⁺) isolated from the indicated strains (isotype and FMO [fluorescence minus one] staining are from Vps34^f/f isolated cells) were analyzed for (A) MHC class I (H-2K^b), (B) MHC class II (H-2I-A/I-E), or (C) CLIP-bound I-A^b expression by flow cytometry. Data are representative of at least two independent experiments (n = 4–7 per genotype). (D) Protein isolated from control and Vps34-deleted C9 cells were analyzed by Western blot for markers of autophagy and vesicle trafficking. β-Tubulin was used as loading control. The graph represents the relative densities of pre-pro-cathepsin L, pro-cathepsin L, and mature cathepsin L bands (CatL) between sgLacZ and sgVps34. Data are representative of two independent experiments. (E) Light micrograph of live control (sgLacZ) and Vps34-deleted (sgVps34) C9 cells (top, scale bar = 100 μm). Thymus tissue sections from 3-d-old Vps34^f/f or Vps34^TEC mice stained with H&E (bottom, scale bar = 75 μm). Arrows highlight areas of significant vacuolization. The graph represents the number of vacuolated areas per medullary islet. Data are from one randomly selected medullary islet from three independent biological replicates of each genotype. (F) sgVps34 and sgLacZ cells were stained with Lysosensor green probe and then analyzed by flow cytometry. The graph represents relative mean fluorescence intensity (MFI) of Lysosensor green. Data are representative of two independent experiments. All data signify the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; nd, none detected by unpaired t test. Source data are available for this figure: SourceData F7.