Figure 3

RR structures induced by inhibitors of CTP/GTP synthesis disassembled in the remaining ≈ 30% of the cells by microinjection of anti-IMPDH2 antibody. COS-7 cells were treated with 1 mM (A) ribavirin, (B) DON, or (C) MPA for 24 h and went under microinjection. Independent of the treatment used to induce RR, disassembly was detected in 76 (29%) of the 263 cells counted (check Figure 2). After 10–15 min of microinjection, images were captured in intervals of 2 min for up to 10 min to document the disassembly of RR. The arrows in A, B, and C indicate RR structures disassembling into pieces. Nuclei were stained with Draq5 (red). In D, E, and F, to evaluate the influence of the microinjection procedure on RR stability, anti-IMPDH2 antibodies or total rabbit IgG were microinjected into ~200 COS-7 cells; after 30 minutes, cells were fixed with 3% paraformaldehyde and stained with anti-IMPDH2 antibody. Control using total rabbit IgG shows no influence on RR structures. Blue color shows DNA labeled by DAPI. Bars, 10 μm. (G) Complete disassembly of RR in cells microinjected with anti-IMPDH2 compared to those microinjected with control total rabbit IgG or neighboring cells not subjected to microinjection. The proportion of cells that show intact RR was decreased in the slide area that we injected anti-IMPDH2 when compared with total rabbit IgG area (p = 0.0001 by Two-tailed Chi-square test).