Fig. 3

TSL2-interacting small molecules increase SMN2 E7 splicing. a RT-PCR of exons 6-to-8 from HeLa cells transfected with SMN^E6−to−8 minigenes¹⁶ and treated with NCI377363 (1, 5 and 10 μM), PK4C9, BJGF466 and 288D (10, 20 and 40 μM) or DMSO (0.04%, controls) for 6, 12 and 24 h (n = 6; three biological and two technical replicates). PK4C9 and BJGF466 elicit maximal E7 inclusion at early time points, which may be due to progressive compound toxicity and/or molecule secretion, as commonly seen in cancer cells. b, c RT-PCR (b) and qRT-PCR (c) showing the dose–response of PK4C9 in GM03813C (SMA) and GM03814B (parental carrier) fibroblasts after 24 h of treatment (n = 6; three biological and two technical replicates). For simplicity, the ladder is shown in a. In RT-PCR, SMN2 isoforms including and excluding E7 are detected simultaneously. In qRT-PCR, SMN2 isoforms including and excluding E7 are amplified in two separate reactions. The dose–response curve of PK4C9 reveals a narrow concentration window but achieves maximal response. Concentrations higher than 50 μM could not be measured due to poor solubility of the compound. d Twenty-four-hour cytotoxicity in GM03814B fibroblasts, using the lactate dehydrogenase (LDH) assay (n = 12; six biological replicates and two technical replicates). PK4C9 showed the lowest toxicity when comparing all molecules at the same concentration (40 μM; left) and the concentration of maximum splicing activity (right; PK4C9 40 μM, BJGF466 40 μM, NCI377363 10 μM and 288D 80 μM). Complete 24-h and 72-h cytotoxicity curves are shown in Supplementary Fig. 8. e RT-PCR from adult D42-Gal4>UAS-SMN2^E6−to−8 Drosophila flies expressing human SMN2^E6−to−8 in motor neurons. Flies were free fed with PK4C9 (25, 50, 100 and 200 μM) or DMSO (0.5%, controls) as larvae. rp49 is the loading control (n = 3 biological replicates of 10–12 individuals each), **p < 0.01. p-values were obtained by applying non-paired, two-tailed t tests with Welch corrections. All graphs represent mean values ± SEM