Figure 3 : 293T-SNCA-3′NL cells having the NanoLuc integration can be used to model deregulated SNCA as seen in sporadic PD.

From: A novel tool for monitoring endogenous alpha-synuclein transcription by NanoLuciferase tag insertion at the 3′end using CRISPR-Cas9 genome editing technique

Figure 3

(a) 293T-SNCA-3′NL cells treated with 10 μM 5-AzadC for 72 hours which induced a significant increase in the NanoLuc activity as compared to the control. The NanoLuc activity was corroborated by increase in α-SYN transcript as shown in the RT-PCR. (b) Methylation status of 23 CpG sites on the SNCA intron1 was determined by bisulfite sequencing. Amplified PCR products were cloned into pGEM-T Easy vector and 10 clones were sequenced. (Left) Comparison of vehicle and 5-AzadC treatment (10 μM, 72 hours) showing unmethylated (open circles) and methylated (closed circles) cytosines for all 10 clones (y-axis) at each of the 23 CpGs in intron1 (x-axis). (Right) Scatter plot showing overall decrease in methylation by 31.7% compared to the control. (c) Similarly, 293T-SNCA-3′NL cells treated with dopamine at 100 μM concentration for 48 hours, increased NanoLuc activity significantly. Increase in the NanoLuc activity was confirmed by RT-PCR after dopamine treatment. (d) Following HDAC inhibitor (sodium butyrate) treatment at concentrations of 2.5 mM and 5.0 mM for 24 hours, the 293T-SNCA-3′NL cells showed a significant dose dependent increase in the NanoLuc activity. This dose-dependent increase in the NanoLuc activity was also confirmed by RT-PCR following same treatment paradigm. β-actin amplification was used as an internal control for all the PCRs. Error bars show the mean from three technical repeats. p values are given for t-test (5-AzadC, dopamine), one-way ANOVA (Sodium butyrate) where *represents p < 0.05, **represents p < 0.01, ***represents p < 0.0001.