Fig. 3
From: A complex of C9ORF72 and p62 uses arginine methylation to eliminate stress granules by autophagy
Symmetric arginine methylation by PRMT5 is required for stress granule clearance by autophagy. a Immunofluorescent microscopy of DDX3 and ubiquitin (Ub) in cells treated with or without arsenite. b Immunofluorescent microscopy of cells labeled with antibodies for symmetric (SYM) or asymmetric (ASYM) dimethylated arginines. c Immunofluorescent microscopy of cells labeled with antibodies for FMRP and proteins containing symmetric (SYM) or asymmetric (ASYM) arginines after treatment with arsenite. d Western blot of total cell lysate and fractions enriched in autophagosomes (Atg fraction#1) and autophagolysosomes (Atg fraction#2) for known autophagy substrates (LC3-II, p62), stress granule proteins (FUS, SMN, TIAR), and symmetrically dimethylated proteins (SYM). e Western blot of immunoprecipitations of proteins containing symmetrically dimethylated arginines from cells expressing HA-p62 (top) or not (bottom); cells were treated with arsenite (+As) or not (−As). f Percentage of cells containing stress granules in cells treated with DMSO (control diluent) or MTA (methylthioadenosine 1 mM, 48 h) after 30 min arsenite treatment, or one hour after removal of arsenite (n = 3, mean ± SD, Student’s t-test). g Representative images of stress granules labeled with TIAR for (f). h Immunofluorescent images of cells labeled with antibodies specific for symmetrically dimethylated arginines (SYM) or FMRP (stress granule marker) in cells treated with arsenite and expressing either control shRNA or PRMT5 silencing shRNA. i Percentage of cells containing stress granules in cells treated with control shRNA or PRMT5 silencing shRNA after 30 min arsenite treatment, or one hour after removal of arsenite (n = 5, mean ± SD, Student’s t-test). j Representative images of stress granules labeled with FMRP for (i). k Left, PLA for endogenous LC3 and FMRP (in SGs) in cells treated with arsenite and transfected with control (Ctl), C9ORF72 (C9), p62, or PRMT5 siRNA; Right, quantification of proximity ligation assay signals (PLA) for LC3 and FMRP in cells under identical conditions to (k) (n = 3, mean ± SD, Student’s t-test). l Western blot control for PLA in (k) in cells treated with control, C9ORF72, p62, or PRMT5 targeting siRNA. HeLa cells were used in all experiments. Scale bar = 10 µm
