Figure 6.

CSN5 downregulation stabilizes OGA, modulating CRT O-GlcNAcylation. (A) A Venn diagram was employed to show the co-expression of O-GlcNAc and ER stress-related genes (https://www.genecards.org/, accessed on May 2023). (B) A2780/DDP cells with or without CSN5 ablation were exposed to cisplatin (20 μM), followed by IP-IB analyses with the indicated antibodies. O-GlcNAcylated CRT and the combination of CRT to OGA were detected. (C) Total proteins of A2780/DDP cells were IP with anti-CSN5 (or anti-CRT) or normal IgG, followed by IB with the indicated antibodies. (D) HEK293T cells were transfected with Flag-CSN5 and HA-OGA in combination. IP-IB analyses were performed with the indicated antibodies after 24 h of transfection. (E) HEK293T cells were transfected with His-CRT and HA-OGA simultaneously. IP-IB analyses were performed with the indicated antibodies after 24 h of transfection. (F, I) DDP cells with or without CSN5 knockdown were exposed to cisplatin (20 μM) for 24 h, and the mRNA levels were determined by qRT-PCR. (G, H, J, K) DDP cells were treated with cycloheximide (CHX, 50 μM) for the indicated time, and the protein half-life time was evaluated by IB and quantified. (L) A2780/DDP cells with or without CSN5 inhibition exposed to cisplatin were treated with MG132 (20 μM) or vehicle control for 6 h before cell lysates, and ubiquitination levels of OGA were evidenced by IP-IB. (M) A2780/DDP cells were exposed to cisplatin (20 μM) for 24 h, and the related protein expression was evaluated by IB and quantified (N). (O) A2780/DDP-shCSN5 cells with or without CSN5 overexpression exposed to cisplatin were treated with MG132 (20 μM) or vehicle control for 6 h before cell lysates, and ubiquitination levels of OGA were evidenced by IP-IB. (P) A2780/DDP-shCSN5 cells with or without CSN5 overexpression were exposed to cisplatin (20 μM) for 24 h, and the related protein expression was evaluated by IB and quantified (Q).