Fig. 3
From: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction
Upregulation of ISGylation through DFMO treatment and downregulation of ISGylation through SPD treatment in the presence of DFMO in A549 cells. (A) Serum deprivation slows down A549 cell growth. A549 cells (2 × 105) are cultured with or without serum in a 6 cm dish, and cell numbers are counted daily. Cell viability is analyzed by trypan blue staining, with stained cells considered dead. (B) DFMO slightly upregulates ISGylation but not ubiquitination. A549 cells are stimulated by hIFN-α with or without DFMO (0.5 or 1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting using an anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. (C) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, as well as that of ISGylation and ubiquitination in (B). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t-test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. (D) SPD partially prevents ISGylation, ubiquitination, UBE1L, UBE2L6, and USP18 expression in the A549 cells. A549 cells are stimulated by hIFN-α with or without SPD (0.5 or 1.0 mM) in the presence of DFMO (1.0 mM) for 2 days. The cell lysates are subjected to immunoblotting with anti-ISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data of three independent experiments. (E) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, and that of ISGylation and ubiquitination in (D). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by human IFN-α is set as 1. Data are expressed as the mean ± standard deviation of three independent experiments. The means of different groups are compared using the Student’s t-test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.
