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. Author manuscript; available in PMC: 2013 Jul 1.
Published in final edited form as: Nat Immunol. 2012 Nov 18;14(1):61–71. doi: 10.1038/ni.2475

Figure 7.

Figure 7

Trex1 regulates lysosomal biogenesis through TFEB and mTORC1. (a) Quantitative RT-PCR analysis of lysosomal and non-lysosomal genes in WT and Trex1−/− MEFs. (b) Fluorescent microscopic analysis of endogenous TFEB localization in WT and Trex1−/− MEFs. Right panel shows percentage of nuclear TFEB in the cell. Averages of 13 cells are shown (error bars, s.d.) *P < 0.05 (Student’s t-test). (c,d) Western blot analysis of TFEB knockdown (c) and qRT-PCR analysis of Ifit1 and Ifit3 mRNA (d) in Trex1−/− MEFs transfected with control or TFEB specific siRNAs. (e) FACS analysis of VSV-PeGFP replication in WT and Trex1−/− MEFs transfected with control or TFEB specific siRNAs for 72 h and mock-infected or infected with VSV-PeGFP for 18 h. *P< 0.05 (Student’s t-test). Data are representative of three independent experiments (error bars, s.d.). (f) Quantitative RT-PCR analysis of Ifit1 expression in WT MEFs transfected with myc-TFEB or pcDNA3 vector plasmid at indicated amount for 24 h. (g) Quantitative RT-PCR analysis of Ifit1 and Mcoln1 in WT MEFs treated with chloroquine at 10, 50 and 100 uM for 16 h. (h, i) Western blot (h) and densitometry (i) analysis of proteins involved in the mTORC1 pathway. WT and Trex1−/− MEFs were uninfected or infected with VSV for 16 h. Densitometry analysis was performed using Image J on 6 independent western blots. WT normalized to 1. *P< 0.05 (Student’s t-test). Data are representative of 6 independent experiments (error bars, s.d.). (j) Quantitative RT-PCR analysis of Ifit1 expression in WT MEFs transfected with indicated siRNAs for 72 h. *P< 0.05 (Student’s t-test). Data are representative of two independent experiments (error bars, s.d.). (k) Western blot analysis of proteins involved in the mTORC1 pathway. WT and Trex1−/− MEFs were transfected with vector or Flag-Trex1 plasmid for 24 h. A representative gel image of 4 independent experiments is shown.