1. Cellular localization of the “writers” and “erasers” in U2OS-G3BP1 cells.
Cellular localization of the “writers” and “erasers” in U2OS-G3BP1 cells. (A) qRT–PCR following combined siRNA knockdown of the “writer complex” (METTL3, METTL14, and WTAP) for 48 h. The levels of each writer mRNA were normalized to the expression of β-actin mRNA. Data are means ± SD (n = 2). Scr denotes scrambled siRNA and accounts for unspecific effects. Representative Western blots (n = 2) to verify the decrease in the expression of each of the proteins in the writer complex using specific antibodies. GAPDH serves as loading control. (B) Localization of METTL3, METTL14, and WTAP under permissive growth and upon stress exposure (500 μM AS, 30 min). G3BP1 detected through its fluorescent GFP tag is used to monitor SG formation under stress. Scale bar, 10 μm. (C) Nuclear localization of FTO (upper panel) and ALKH5 (bottom panel) “erasers” in both control growth and following treatment with 500 μM AS for 30 min. G3BP1 detected through its fluorescent GFP tag is used to monitor SG formation under stress. Scale bar, 10 μm. (D) Total RNA isolated from equal amount of HEK-TIA1 cells grown at permissive (control) conditions or exposed to various AS concentrations and detected with m6A antibody or methylene blue (MB). Source data are available for this figure.
Maximilian Anders et al. LSA 2018;1:e
© 2018 Ignatova et al.