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. 2020 Mar 5;15(3):e0230156. doi: 10.1371/journal.pone.0230156

Fig 4. TJ-35 suppresses dephosphorylation of ULK1 and TFEB specifically among mTORC1 substrates.

Fig 4

A. GFP-TFEB expressing HeLa cells were treated with or without TJ-35 for 4 h, and then shifted to DMEM or EBSS with or without TJ-35 for 2 h. Bar represents 10 μm. The graph shows quantification of GFP-TFEB that localized in the cytoplasm or nucleus. Percent of cells with cytoplasmic, nucleus or both. We counted 30 cells in three independent experiments. B. HeLa cells were treated with or without TJ-35 in DMEM or EBSS for 4 h, and then total RNA was extracted. Expression levels of ATPV6 and LAMP1 versus GAPDH were monitored by RT qPCR. The graph shows fold change. CE. HeLa cells were treated with or without TJ-35 for 4 h, shifted to DMEM or EBSS with or without TJ-35 or Torin-1 for 2 h, and then subjected to immunoblotting with anti-TFEB, anti-phosopho-S6K, anti-p70 kinase, anti-4E-BP1 and anti-Tubulin. The samples of C and E were derived from the same preparation. D. HeLa cells were treated with or without TJ-35 for 4 h, shifted to DMEM or EBSS with or without TJ-35 or Torin-1 for 2 h, and then subjected to immunoblotting with anti-phospho-ULK1(Ser757), anti-ULK1 and anti-Tubulin.