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[Preprint]. 2023 Jan 10:rs.3.rs-2395172. [Version 1] doi: 10.21203/rs.3.rs-2395172/v1

Figure 5: Characterization of R12–8–44–3 in a reporter cell line.

Figure 5:

(A) Chemical structures of R12–8–44–3 and inactive negative control compound R12–8–32 (aka “R12-neg”). As a positive control, Ro 08–2750 (aka “Ro”) is also included in these studies. (B) Schematic of reporter assay. HEK293 cells are transfected with a plasmid encoding an arbitrary gene (Luc) with UAG repeats (to recruit MSI2) and MS2 repeats (for pulldown) appended to its 3’ UTR. The same vector also includes FLAG-labeled MS2-BP. Upon anti-FLAG immunoprecipitation (IP), the FLAG-labeled MS2-BP allows purification of the Luc mRNA along with any associated factors. (C) Prior to IP, cells express Luc mRNA to a similar extent irrespective of whether they were treated with DMSO, R12-neg, R12–8–44–3, or Ro. (D) Prior to IP (“input”), MSI2 is detected in all samples to a similar extent (as is KU-86 loading control). After pulldown of Luc mRNA, MSI2 is found associated with the mRNA in cells treated with DMSO or R12-neg, but not in cells that had been treated with R12–8–44–3 or Ro. Representative image is shown; quantification is plotted as the average of 2 independent experiments ± S.E.M. Statistical analysis was performed using unpaired two tailed t-test, with * indicating p<0.05. Uncropped Western blots from this experiment are provided as Figure S8.