Forming cytoophidia prolongs the half-life of CTP synthase

a HEK-293T cells stably expressing mCTPS1-GFP were treated with 4？μg/ml DON for the indicated time, and cell lysates were prepared and analyzed by immunoblotting with anti-GFP antibody. b Quantitative data of mCTPS1-GFP protein level in a. c, e SW480 cells stably expressing mCTPS1WT-GFP, mCTPS1H355A-GFP (c) or mCTPS1R294D-GFP (e) were cultured for 3 days, and then subjected to western blotting analysis with anti-GFP antibody. d, f Quantitative data of mCTPS1-GFP protein level in c and e, respectively. g–i SW480 cells expressing endogenous wild-type CTPS1 (CTPS1WT) and those expressing R294D mutant CTPS1 (CTPS1R294D) were cultured for 3 days, followed by immunostaining (g) or western blotting against CTPS1 (h). i Quantitative data of CTPS1 protein levels in h. j The CTPS1 mRNA levels of SW480 CTPS1WT and SW480 CTPS1R294D+/？ cells were analyzed by quantitative reverse transcription PCR (qRT-PCR). k Schematic diagram of TRE3G Tet-On system. l, m HEK-293T cells stably expressing Tet-On mCTPS1-GFP were cultured in the medium containing doxycycline (200？ng/ml) for 24？h, followed by culturing with the doxycycline-free medium without (l) or with (m) DON (4？μg/ml) for the indicated time. Lysates were prepared and analyzed by immunoblotting with appropriate antibodies. n Quantitative data of mCTPS1-GFP protein levels in l and m. o HEK-293T cells stably expressing Tet-On mCTPS1-GFP were cultured in the medium containing doxycycline (200？ng/ml) for 24？h, followed by culturing with the doxycycline-free medium with the indicated concentration of DON for 48？h. Lysates were prepared and analyzed by immunoblotting with appropriate antibodies. p HEK-293T cells stably expressing Tet-On mCTPS1WT-GFP (left three lanes) or Tet-On mCTPS1H355A-GFP (right three lanes) were treated with doxycycline (200？ng/ml) for 24？h, followed by culturing with doxycycline-free medium with the indicated concentration of DON for 48？h. Anti-GFP antibody was used to detect mCTPS1-GFP levels. q Quantitative data of mCTPS1-GFP protein levels in p. r–t SW480 cells stably expressing Tet-On mCTPS1WT-GFP (r), mCTPS1R294D-GFP (s) or mCTPS1H355A-GFP (t) were grown in the medium containing doxycycline (200？ng/ml) for 24？h, followed by culturing with the doxycycline-free medium for the indicated time. Lysates were prepared and subjected to western blotting analysis with anti-GFP antibody. u Quantitative data of mCTPS1-GFP protein levels in r–t. v HEK-293T cells stably expressing mCTPS1-GFP were transfected with HA-Ubiquitin, and then treated with the indicated concentration of DON for 36？h. MG132 (20？μM)