Fig. 6: HeLa cells overexpressing expanded C9orf72 repeats display spontaneous SGs and decreased PML-NB numbers.

A HeLa Kyoto cells were transfected for 24 h with a vector coding for 66 repeats (66 R). Then cells were fixed, subjected to FISH to visualize RNA foci and to immunostaining with antibodies specific for the SG protein G3BP1 and for ubiquitin (clone FK1). Nucleic acid was labeled using DAPI. Scale bar is 10 μm. The percentage of 66 positive (66 +) and 66 negative (66 -) cells with SGs is shown; n = 4, −/+ s.e.m. Number of cells quantified: 66 R - (1243); 66 R + (818); Student’s t-test. B HeLa Kyoto cells were transfected for 24 h with an empty vector (0 R), or vectors coding for 2 or 66 repeats (2 R, 66 R). Cells were fixed, subjected to FISH to visualize RNA foci and to immunostaining with antibodies specific for polyubiquitin (FK1). Nucleic acid was labeled using DAPI. Scale bar is 10 μm. The quantification of the percentage of cells with cytoplasmic polyubiquitin-positive foci is shown. n = 5, −/+ s.e.m. Number of cells quantified: 0 R (430); 2 R (1491); 66 R (762). One-way ANOVA, Post-hoc Bonferroni-Holm test. C-E: HeLa Kyoto cells were transfected for 24 h with an empty vector (0 R), or vectors coding for 2 or 66 repeats (2 R, 66 R). Then cells were either treated with OP-puro (25 μg/ml) alone or with MG132 (20 μM) for 3 h. Cells were fixed, subjected to FISH to visualize RNA foci and to immunostaining with antibodies specific for PML and polyubiquitin (FK1). The white arrowheads show polyubiquitin-positive cytoplasmic aggregates. Nucleic acid was labeled using DAPI. Scale bar is 10 μm. Representative images are shown in (C). D, E The average number of PML-NBs in 0 R, 2 R and 66 R expressing cells shown in (C). n = 3, −/+ s.e.m. Number of cells quantified in (D): 0 R (588); 2 R (567); 66 R (390). Number of cells quantified in (E): 0 R (482); 2 R (613); 66 R (442). One-way ANOVA, Post-hoc Bonferroni-Holm test.