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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Neuron. 2021 Oct 12;109(23):3758–3774.e11. doi: 10.1016/j.neuron.2021.09.031

Figure 6. TRIP12 Knockdown Rescues α-syn PFFs-induced Neurodegeneration in Both Control hiPSC- and GCase N370S PD iPSC-derived DA Neurons.

Figure 6

(A-M) Control iPSC (C1) and GBA1-PD hDA neurons (GBA1-PD), respectively, were transduced with the lentivirus expressing either control shRNA or TRIP12 shRNA at three days before α-syn PFFs treatment.

(A) Fluorescent analysis was conducted in 5 μg/ml α-syn PFFs-treated hDA neurons for ten days using anti-p-α-syn and TH antibodies.

(B) In α-syn PFFs-treated hDA neurons, p-α-syn level was quantitated and shown as bar graphs (n=3, each group).

(C-J) hDA neurons were sequentially fractionated in TX-soluble followed by TX-insoluble buffer.

(C and D) Western blot analysis of TX-soluble and TX-insoluble fractions using the indicated antibodies.

(E-H) Quantitation of the levels of TRIP12, GCase, TH, and α-syn in TX-soluble fraction and shown as bar graphs (n=3, each group).

(I and J) Quantitation of α-syn and pS129-α-syn in TX-insoluble fraction (n=3, each group).

(K) GCase activity in total fractions (n=3, each group).

(L) GBA1 mRNA level in total fractions (n=3, each group).

(M) α-syn PFFs-induced cytotoxicity was evaluated using LDH assay in culture media (n=3, each group). All quantitated data shown in bar graphs were conducted in both α-syn PFFs-treated control hiPSC and GBA1-PD hiPSC DA neurons. Data are represented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).