Effect of ASCC2, ASCC3 or HBS1L knockdowns on general translation and PF8503-induced stalling.
(A) Metabolic labeling of ongoing translation during 30 min treatment of K562_dCas9-KRAB cells expressing scrambled sgRNA, with PF8503 (7.5 μM), DMSO control, or cycloheximide (100 μg/mL). Shown is IRDye800 labelled L-AHA incorporated into newly synthesized proteins of a representative experiment carried out in duplicate. (B) L-AHA incorporation in newly synthesized proteins in K562_dCas9-KRAB cells expressing scrambled, ASCC2, HBS1L, or ASCC3 sgRNA during 30 min treatment with DMSO or PF8503 (7.5 μM). Ratio of L-AHA incorporation for each knock-down relative to the control cell line are normalized to total protein ratio, determined by Bradford assay. Experiments were carried out in duplicate with the mean and standard deviation shown. (C) Reporter mRNAs used for cell-based assays. ARCA, m7G cap with m7G nucleotide 3’-O-methylated. 5’-UTR HBB, 5’-untranslated region of the HBB gene. PCSK9(1–35), codons 1–35 of the PCSK9 gene. (D) Inhibition of the PCSK9(1–35) reporter mRNA in K562-dCas9-KRAB cells expressing scrambled, ASCC2, HBS1L, or ASCC3 sgRNA, after 6–8 hr treatment with DMSO or various concentrations of PF8503. Experiments were carried out in biological triplicate, with mean and standard deviations at each PF8503 concentration shown.