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. 2019 Aug 22;4(16):e129719. doi: 10.1172/jci.insight.129719

Figure 3. Sustainable TFEB transgene expression in vitro and in vivo.

Figure 3

(A) Representative images (top) and quantification (bottom) of TFEB and HA tag immunoblotting in HEK293T cells transfected for 48 hours with the mTFEB-expressed plasmid under the neuronal CMVie/Synapsin promoter (CMVie/Synapsin-mTFEB-HA-WPRE). (B) Representative images (top) and quantification (bottom) of TFEB and Flag tag immunoblotting in HEK293T cells transfected for 48 hours with the mTFEB-expressed plasmid under the oligodendroglial promoter MBP (MBP-mTFEB-3×Flag-WPRE). Data represent mean ± SEM. Comparisons were made using nonparametric t test. *P < 0.05 compared with nontransfected cells. (C) Confocal images using HA tag and tyrosine hydroxylase (TH) antibodies in the ipsilateral SN of mice injected with the CMVie/Synpasin-mTFEB-HA virus 5 months after the injection. The white arrows indicate the presence of HA-tagged mTFEB signal into the nucleus of TH-positive neurons. Scale bar: 10 μm. (D) Representative images (top) and quantification (bottom) of HA tag immunoblotting in the ipsilateral SN of CMVie/Synapsin-mTFEB-HA–injected WT and PLP mice 5 months after the injection. n = 5 per group. Data represent mean ± SEM. Comparisons were made using 1-way ANOVA and Tukey’s correction for multiple comparisons. White bars, control; blue bars, CMVie/Synapsin-mTFEB-HA. (E) Confocal images using Flag tag and CNPase antibodies in the ipsilateral SN of mice injected with the MBP-mTFEB-3×Flag virus 5 months after the injection. The white arrow indicates the presence of Flag tag signals into the nucleus of CNPase-positive oligodendrocytes. Scale bar: 10 μm. (F) Representative images (top) and quantification (bottom) of Flag tag immunoblotting in the ipsilateral SN of MBP-mTFEB–injected WT and PLP mice 5 months after the injection. Data represent mean ± SEM. Comparisons were made using 1-way ANOVA and Tukey’s correction for multiple comparisons. White bars, control; green bars, MBP-mTFEB-3×Flag. In A, B, D, and F, lanes were run on the same gel but were noncontiguous.