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. 2018 Aug 24;14(8):e1007592. doi: 10.1371/journal.pgen.1007592

Fig 7. Mammalian KCTD11 is a negative regulator of mTORC1 in response to low amino acids.

Fig 7

(A) Immunoblots for yeast TORC1 activity (anti-pS6) and Pgk loading control in whi2 knockout yeast expressing N-terminal HA-tagged proteins, yeast Whi2, human KCTD11 (hK11), hKCTD7 (hK7), and mouse KCTD8 (mK8) proteins expressed from PGK promoter-driven plasmids before and after switching from control/high (SCCSH) to low amino acids (SCME). Expressed proteins are detected with anti-HA antibody of the same lysates. *Presumed non-specific band. (B) The activity of mTORC1 in monkey kidney COS7 cells was assessed on immunoblots for phospho-S6K relative to total S6K in cells expressing HA-/FLAG-tagged mammalian KCTD proteins or empty vector harvested after 50 min of amino acid and serum deprivation with and without readdition of amino acids for 10 min. Representative of 3 independent experiments is shown. (C) As described in panel B for hKCTD11. Quantification of mTORC1 activity was determined as the ratio of phospho-S6K to total S6K for 3 independent experiments and presented as mean ± SD directly below corresponding lanes as labeled for immunoblots. Two-tailed t-test for cells transfected with HA-hKCTD11 versus empty vector after amino acid withdrawal, p = 0.047, and after readdition of amino acids, p = 0.003. (D) Endogenous KCTD11-dependent mTORC1 activity was analyzed in human HEK293 cells transfected with scrambled control and hKCTD11 shRNAs for 48 h followed by amino acid and serum deprivation for 50 min with and without readdition of amino acids for 10 min. Immunoblots for both phospho-S6K relative to total S6K (direct mTORC1 target) and for phospho-S6 relative to total S6 were quantified for 4–8 independent experiments and presented as mean ± SD directly below corresponding lanes as labeled for immunoblots. Two-tailed t-test comparing amino acid-treated samples for shRNA2 versus control shRNA (n = 8), p = 3.7 x 10−4 (p-S6K/S6K), p = 5.4 x 10−5 (p-S6/S6), and comparing shRNA2 versus ineffective shRNA1 (n = 4), p = 0.016 (p-S6K/S6K), p = 1.2 x 10−3 (p-S6/S6). (E) KCTD11 shRNAs were evaluated on immunoblots for their ability to suppress the expression of HA-tagged KCTD11 when co-transfected in human HEK293 cells for 24 h and grown under normal conditions.