EWS-Fli-1
turns over by a lysosome-dependent mechanism: (A) Knockdown
of CIMPR or VPS26A results in destabilization of FLAG-EWS-Fli-1. 293
cells were cotransfected with FLAG-EWS-Fli-1 and shRNA against luciferase
(control), CIMPR, or VPS26A. Forty-eight hours after transfection,
the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting.
Nucleolin serves as a loading control. (B) TFEB induces EWS-Fli-1
degradation in 293 cells. 293 cells were cotransfected with FLAG-EWS-Fli-1
and HA-TFEB or empty vector. Forty-eight hours after transfection,
the levels of FLAG-EWS-Fli-1 were examined by anti-FLAG immunoblotting.
Tubulin serves as a loading control. (C) Chloroquine stabilizes EWS-Fli-1
in 293 cells. 293 cells were transfected with FLAG-EWS-Fli-1. Transfected
cells were left untreated (control) or treated with 100 μM chloroquine
for 12 h. The levels of FLAG-EWS-Fli-1 were examined by anti-FLAG
immunoblotting. Tubulin serves as a loading control. (D) Cathepsin
D degrades EWS-Fli-1, but not p53, in 293 cells. 293 cells were cotransfected
with FLAG-EWS-Fli-1 and cathepsin D or empty vector. Transfected cells
were left untreated or treated with 100 μM chloroquine for 12
h or 100 nM pepstatin A for 12 h. 293 cells were cotransfected with
FLAG-p53 and cathepsin D or empty vector. The levels of FLAG-EWS-Fli-1
and FLAG-p53 were examined by anti-FLAG immunoblotting. Nucleolin
serves as a loading control. (E) Cathepsin D degrades endogenous EWS-Fli-1
in A673 Ewing sarcoma cells. A673 cells were infected with a lentivirus
vector expressing cathepsin D or an empty vector, the infected cells
were selected with puromycin, and the levels of endogenous EWS-Fli-1
were examined by anti-Fli-1 C-terminus antibody immunoblotting at
4 days after infection. Nucleolin serves as a loading control. (F)
Chloroquine stabilizes endogenous EWS-Fli-1 in A673 cells. A673 cells
were left untreated, treated with 100 μM chloroquine for 12
h, or treated with 10 μM MG-132 for 12 h. The levels of EWS-Fli-1
were examined by anti-Fli-1 C-terminus immunoblotting. While chloroquine
increased the levels of endogenous EWS-Fli-1, MG-132 had no effect
on the EWS-Fli-1 protein levels, suggesting that EWS-Fli-1 turns over
by a lysosomal, but not proteasomal mechanism. (G) Endogenous EWS-Fli-1
in A673 cells displays increased lysosomal location upon chloroquine
treatment. A673 cells were treated with 100 μM chloroquine for
12 h or left untreated, and the whole cell extract (WCE) and lysosomal
fraction were isolated. The abundance of EWS-Fli-1, LAMP2, p62/SQSTM1,
and mSin3A in each fraction was determined by immunoblotting.