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. 2015 Dec 1;4(12):e266. doi: 10.1038/mtna.2015.38

Figure 3.

Figure 3

HTT10150 shows dose-dependent silencing of huntingtin by passive uptake in primary neurons. (a) Huntingtin mRNA levels in primary striatal (black) or cortical (gray) neurons 1 week after treatment with the indicated concentrations of HTT10150. Huntingtin mRNA levels were normalized to Ppib mRNA. Data are expressed as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (1.25 µmol/l). (b) Huntingtin protein levels in primary neurons 1 week after treatment with the indicated concentrations of HTT10150. Huntingtin and β-tubulin proteins were quantified by densitometry of western blots, and huntingtin protein levels were normalized to β-tubulin. Data are expressed relative to the level of huntingtin protein in untreated control cells. (n = 5 neuronal preparations from separate pups, mean ± SD). NTC, nontargeting control (1.25 µmol/l). Graph of silencing in primary cortical neurons after 1 week is representative, results confirmed in five separate experiments. (c) Original western blots from graph in b. Primary cortical neurons were cultured from five individual pups (#1–5) and incubated with HTT10150 at concentrations shown for 1 week. Huntingtin protein levels were detected by western blot using antibody AB1 (Huntingtin 1–17). NTC, nontargeting control. (d) Primary neurons were incubated with HTT10150 at concentrations shown, for 1, 2, and 3 weeks. Level of huntingtin mRNA was measured using QuantiGene (Affymetrix) normalized to housekeeping gene, Ppib (cyclophillin B), and presented as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (1.5 µmol/l). Graph of silencing in primary cortical neurons after 1 week is representative, results confirmed in five separate experiments. (e) Primary cortical neurons were incubated with two different Htt hsiRNA sequences HTT10150 and HTT10146 at concentrations shown for 72 hours. Level of huntingtin mRNA was measured using QuantiGene (Affymetrix) normalized to housekeeping gene, Ppib (cyclophillin B), and presented as percent of untreated control (n = 3 wells, mean ± SD). NTC, nontargeting control (1.5 µmol/l). Graph of HTT10150 silencing in primary cortical neurons after 72 hours is representative, results confirmed in seven separate experiments.