Fig. 2 |. in vitro ASO screen.

a, Assessment of the specificity of ASOs and their effects on progerin, lamin A and lamin C mRNA by ddPCR. ASOs were transfected into immortalized HGPS cells and analyzed 24–48 h after transfection (Methods). The data were normalized to housekeeping gene TBP and expressed as relative to control. Data are mean ± s.d. from three experiments. Data for representative ASOs indicated in Fig. 1., Box A, are shown. b, ASO chemistry significantly affects ASO potency. ASOs with the same sequence were synthesized in three different chemistries (top) and tested in HGPS cells (Methods). Levels of lamin A, progerin and lamin C mRNA were quantified by ddPCR, and the data were normalized to housekeeping gene TBP and expressed as relative to control (bottom). Data are mean ± s.d. from three experiments. c, Elimination of ‘G-quartets’ results in highly effective oligos. Inosine-substituted ASOs were tested in HGPS cells, and levels of human lamin A, progerin and lamin C mRNA levels were quantified by ddPCR (Methods). The data were normalized to housekeeping gene TBP and expressed as relative to control. Data are mean ± s.d. from three experiments. Data for representative ASOs indicated in Fig. 1., Box B, are shown. d, Western blot analysis. Total protein extracts were prepared from HGPS cells transfected with inosine ASOs, and the lamin A isoforms (lamin A, progerin and lamin C) were detected using mouse anti-lamin A/C antibody (Methods). The data are representative of two experiments, are normalized to housekeeping β-actin and are expressed as relative to control.