Figure 5 .

FOXO1 requires mRNA binding proteins to affect polyQ levels. (A) GO analysis (using DAVID 6.8) of proteins bound to GFP-HTT^CAG47mRNA with (black bars) or without (grey bars) Flag-FOXO1 overexpression. (B) qPCR analysis of STAU1, IGF2BP3, FUS, DDX18, DDX41 and TAF15 in cells that express Flag-FOXO1 or not, and in presence or absence of HTT^Q71GFP. All data were normalized to GAPDH as reference and were corrected to EV. (C) Representative immunofluorescence pictures detecting p-bodies (using DDX6 antibodies). HEK293T cells expressing HTT^Q25GFP (Left panel) or HTT^Q71GFP (Right panel) with and without Flag-FOXO1 (lower and upper row) were stained with a DDX6 antibody (red). Nucleus stained with Hoechst (blue). (D) Graph depicting quantification of the number of p-bodies per cell of cells treated as in C. (E) Graph depicting p-bodies intensity of cells treated as in C. (D and E) Mean and SEM of three independent experiments are shown. P-values were derived from two-tailed Student’s t test. (F) Filter trap analysis of cells expressing HTT^Q71GFP, Flag-FOXO1 (or not) after knock down of STAU1, IGF2BP3 or DDX18 using CRISPRi. Lower panel depicts a filter trap probed with GFP antibodies. Upper panel depicts the graph with the mean and SEM of three independent experiments. (G) qPCR analysis of cells treated as in F. Relative quantification of HTT^CAG71GFP mRNA expression normalized to GAPDH and corrected to EV. P-values were derived from 1 way analysis of variance with the Bonferroni correction. ^*P < 0.05; ^**P < 0.001; ^***P < 0.0001