Journal: Cell reports
Article Title: A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons.
doi: 10.1016/j.celrep.2025.115695
Figure Lengend Snippet: Figure 4. Unfolded protein response inhibitor periplocin reduces DPR turnover through activation of ERK1/2 (A) Concentration-dependent increase in endogenous polyGA-HiBiT levels upon periplocin treatment for 3 days in the DPR-HiBiT assay (presented as fold change over DMSO control, **p ≤0.01, ****p ≤0.0001, one-way ANOVA, N = 3 inductions). Half-maximal effective concentration (EC50) is 0.397 μM (non-linear regression). (B) Endogenous polyGA levels after 3 days of periplocin treatment at EC50 (0.397 μM) and EC97.5 (1.25 μM) in neurons of 3 independent patient iPSC lines, measured by MSD immunoassay (*p ≤0.05, one-way ANOVA, N = 3 unedited cell lines). (C) Overview of live-DPR labeling protocol: a doxycycline-inducible LgBiT expression cassette was introduced in i3N DPR-reporter cells by lentiviral transduction. Cells are induced into neural progenitor cells by doxycycline treatment while simultaneously triggering LgBiT expression for labeling of DPR-HiBiT proteins. After baseline luminescence detection (0 h), cells are treated in absence of doxycycline to determine reduction of labeled DPRs over time. (D) Live labeling of endogenous polyGA-HiBiT enables determination of turnover. Degradation of endogenous polyGA-HiBiT is slowed by periplocin treatment (***p ≤0.001, ****p ≤0.0001, two-way repeated measures ANOVA, N = 3 inductions). (E and F) (E) Western blot and (F) quantification of the phosphorylated to total ERK1/ERK2 ratio in i3N GA-HiBiT neurons after 3 days of treatment with periplocin at EC50 (0.397 μM) and EC97.5 (1.25 μM) doses (**p ≤0.01, ***p ≤0.001, one-way ANOVA, N = 3 inductions). (G and H) (G) Western blot and (H) quantification of the phosphorylated to total ERK1/ERK2 ratio in i3N GA-HiBiT neurons after 6 days of treatment with the MEK1/ 2 inhibitor trametinib (0, 0.01, 0.1, 1 μM), followed by an additional 3 days in combination with either DMSO or the EC50 dose of periplocin (***p ≤0.001, one-way ANOVA, N = 3 inductions). (I) Quantification of polyGA-HiBiT luminescence under the same experimental conditions as described in (G) and (H) (**p ≤0.01, ***p ≤0.001, one-way ANOVA, N = 3 inductions). Data are presented as mean ± SD unless indicated otherwise.
Article Snippet: Transfection was performed with 14.1 μg of 2nd generation lentiviral packaging plasmid PAX (Addgene #12260), 9.36 μg of VSV-G envelope expressing plasmid (Addgene #12259), and 14.1 μg of the lentiviral transfer plasmid of interest, using Lipofectamine 3000 Transfection Reagent (Invitrogen) following the manufacturer’s instructions.
Techniques: Activation Assay, Concentration Assay, Control, Labeling, Expressing, Transduction, Western Blot
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