Figure 3.

VCP blocks TOLLIP-mediated autophagic degradation of TMEM63A. (A-B) MDA-231 and BT549 cells were treated with or without 200 nM Baf-A1 (A) or 20 mM NH₄Cl (B) for the indicated times, and then subjected to immunoblotting analysis with the indicated antibodies. (C) LM2-4175 and Hs578T cells were treated with or without 1 μM rapamycin (Rapa) for the indicated times, and then subjected to immunoblotting analysis with the indicated antibodies. (D) MDA-231 and BT549 cells were treated with or without 1 mM 3-MA for the indicated times, and then subjected to immunoblotting analysis with the indicated antibodies. (E) MDA-231 and BT549 cells were transfected with siNC and two independent siRNAs targeting ATG5. After 48 h of transfection, total cellular lysates were subjected to immunoblotting analysis with the indicated antibodies. (F) HEK293T cells were transfected with empty vector pLVX and Flag-TMEM63A, and were subjected to IP and immunoblotting analysis with the indicated antibodies after 48 h of transfection. (G) Schematic presentation of functional domains of TOLLIP [40]. LIR, LC3-interacting region; CUE, coupling of ubiquitin conjugation to ER degradation domain; C2, C2 domain. (H) HEK293T cells were transfected with the indicated expression vectors, and then subjected to IP and immunoblotting analysis with the indicated antibodies after 48 h of transfection. (I) MDA-231 and BT549 cells stably expressing pLVX and HA-TMEM63A were subjected to IP and immunoblotting analysis with the indicated antibodies. (J) LM2-4175 and Hs578T cells stably expressing pLVX and HA-TOLLIP were subjected to immunoblotting analysis with the indicated antibodies. (K) MDA-231 and BT549 cells were transfected with siNC and two independent siRNAs targeting TOLLIP. After 48 h of transfection, total cellular lysates were subjected to immunoblotting analysis with the indicated antibodies. (L) LM2-4175 and Hs578T were treated with the or without VCP inhibitor CB-5083 alone or in combination with Baf-A1 for 8 h and then subjected to immunoblotting analysis with the indicated antibodies. (M) HEK293T cells stably expressing empty vector pLVX and Flag-TMEM63A were treated with or without 3 μM VCP inhibitor CB-5083 for 4 h, and then subjected to IP and immunoblotting analysis with the indicated antibodies. (N) MDA-231 and BT549 cells stably expressing Flag-TMEM63A and HA-TOLLIP were treated with DMSO or 3 μM VCP inhibitor CB-5083 for 4 h. Immunofluorescence staining was performed with an anti-Flag (red) or anti-HA (green) antibodies. DNA was counterstained with DAPI (blue). Typical colocalization between Flag-TMEM63A and HA-TOLLIP (yellow) is indicated by white arrows (scale bar: 7.5 μm). In panels A-E, J-M, densitometric quantitation of Western blots was performed using ImageJ.