Journal: Nucleic Acids Research

Article Title: High-throughput screen of 100 000 small molecules in C9ORF72 ALS neurons identifies spliceosome modulators that mobilize G4C2 repeat RNA into nuclear export and repeat associated non-canonical translation

doi: 10.1093/nar/gkaf253

Figure Lengend Snippet: SML compounds increase DPR levels and enhance DPR toxicity, measured by cell proliferation. ( A ) Schematic representation of the Dox-inducible RAN reporter construct used for stable insertion into HEK293 cells, including an upstream Stop codon, no ATG and fusing three reporter tags in different frames downstream of the repeat. Depending on the reading frame, polyGA DPRs carrying a Myc-tag, polyGP DPRs carrying an HA-tag, or polyGR DPRs carrying a FLAG-tag are expressed upon Dox induction of reporter transcription. ( B ) Widefield fluorescent microscopy images showing expression of the different DPRs (Myc-tag: polyGA; HA-tag: polyGP; Flag-tag: polyGR) in RAN reporter lines upon Dox induction, showing high expression of polyGA and polyGP DPRs, and very low expression of polyGR. Zoomed-in confocal figures (lower panel) show subcellular localization of DPR inclusions in the HEK293 152xG4C2 reporter lines, with mostly nuclear localization with some cytoplasmic aggregates for polyGA and polyGP. PolyGR showed mostly cytoplasmic localization ( C ) Quantification of the images represented in Fig. . Each datapoint is an average of 6 wells, with approx. 5′000 cells per well. Statistical differences are shown (t test; **** P < 0.0001** P = 0.002). ( D ) Confocal images of polyGA and polyGP in HEK293 RAN reporter cells treated with 100 nM of SML-3 and PlaB for 24h, showing an increased production of DPRs induced by SML-3 and PlaB compared to Dox only or Dox + SML-4. Zoomed-in frame (Dox panel) of PlaB + Dox treated HEK293 RAN reporter cells suggests nucleolar polyGA inclusions, consistent with previous reports . ( E ) Cell proliferation measured by continuous bright field imaging (Incucyte) for RAN reporter cells with and without Dox-induced RAN expression and 100 nM SML-3, SML-4 or PlaB treatment for up to 100 h ( n = 8 wells and error bars are SD). SML-3 and PlaB treatment significantly enhanced the inhibitory DPR effect on cell proliferation, showing that increased levels of DPRs are inducing reduced cell proliferation (t test, P < 0.0001).

Article Snippet: Primary antibodies were incubated for 3 h or overnight at 4°C in blocking solution: α-Myc tag (Thermo, MA1-980, 1:250); α-HA tag (CellSignaling, #3724, 1:1600); α-Flag tag (Sigma, F3165, 1:400); α-polyGA (Millipore, MABN889, 1:500); α-polyGP (Proteintech, 24494–1-AP, 1:250).

Techniques: Construct, FLAG-tag, Microscopy, Expressing, Imaging