Activated Dectin-2 and Dectin-3 are sorted into lysosomes by the ESCRT system for degradation. (A) Hyphae-induced degradation of Dectin-2 and Dectin-3 in mouse BMDMs, which were pretreated with or without proteasome inhibitor (MG132) or lysosome inhibitor (chloroquine). Cell lysates were probed with the indicated antibodies. (B) Hyphae-induced degradation of Dectin-2 and Dectin-3 in iBMDMs, which were transfected with siRNA against ESCRT-0 subunits (STAM and HRS), ESCRT-I subunits (Vps28 and Tsg101), and nontargeting control siRNA using Trans–IT-TKO transfection reagent (Mirus). (C) Nuclear p65 in iBMDMs, which were transfected with siRNA against ESCRT-0 subunits (STAM and HRS), ESCRT-I subunits (Vps28 and Tsg101), and nontargeting control siRNA before stimulation with C. albicans hyphae (MOI = 0.1) for the indicated times. (D) ELISA results for TNF, IL-6, and IL-12p40 in supernatants of iBMDMs, which were transfected with siRNA against ESCRT-0 subunits (STAM and HRS), ESCRT-I subunits (Vps28 and Tsg101), and nontargeting control siRNA before stimulation with C. albicans hyphae (MOI = 0.1) for 12 h. (E) STAM protein amounts in WT and STAM-deficient iBMDMs. The ESCRT-0 subunit STAM gene was disrupted in iBMDM cells using the CRISPR/Cas9 system. (F–H) Nuclear p65 in WT and STAM-deficient iBMDMs upon stimulation with 40 µg/ml of precoated α-mannans (F), 40 µg/ml LAM (G), or 50 µg/ml TDM (H) for the indicated times. (I) ELISA results for TNF in supernatants of WT and STAM-, HRS-, and Vps28-deficient iBMDMs upon stimulation with 40 µg/ml of precoated α-mannans, 40 µg/ml LAM, or 50 µg/ml TDM for 12 h. (A–C and E–H) The data shown are representative of three independent and reproducible experiments. (D and I) Data are means ± SD of triplicate samples and are representative of three independent experiments. *, P < 0.05; **, P < 0.01 (Student'’s t test or ANOVA). Ctl, control; PCNA, proliferating cell nuclear antigen; Usti., unstimulated; Vec, vector.