Fig. 1
From: Spermidine reduces ISGylation and enhances ISG15–USP18 interaction
Downregulation of ISGylation and UBE2L6, but not of UBE1L, Herc5, and USP18 through SPD treatment. (A) SPM and SPD partially prevent ISGylation and UBE2L6 expression in MCF10A cells. MCF10A cells are stimulated with hIFN-α with or without SPM or SPD (100, 200, or 400 µM) for 2 days. The cell lysates are subjected to immunoblotting with anti-hISG15, anti-ubiquitin, anti-UBE1L, anti-UBE2L6, anti-Herc5, or anti-USP18 antibody. Ponceau S staining is used as a loading control. Representative data from three independent experiments. (B) Quantification of UBE1L, UBE2L6, Herc5, and USP18 expression levels, and that of ISGylation and ubiquitination from (A). 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 not significant (N.S.), whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. (C) Serum deprivation slows down MCF10A cell growth. MCF10A cells (3 × 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. (D) SPM and SPD partially prevent ISGylation in RAW264.7 cells. RAW264.7 cells are stimulated by LPS with or without SPM or SPD (200, 400, or 800 µM) for 2 days. The cell lysates are subjected to immunoblotting with an anti-mouse ISG15 antibody. Ponceau S staining is used as a loading control. Representative data from five independent experiments. (E) Quantification of ISGylation in (D). Each signal is normalized to that of Ponceau S staining. The signal intensity of cells stimulated by LPS 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.
