Extended Data Fig. 2: Intracellular valine abundancy dictates subcellular distribution of HDAC6.
From: Human HDAC6 senses valine abundancy to regulate DNA damage
a, The negligible effect on the acetylation of tubulin upon valine deprivation for 6 hrs. b, Knockdown of HDAC6 in HCT116 cells has little effect on the mTOR signalling with or without valine deprivation. c, Effects of BCAA (valine, leucine and isoleucine) on the subcellular localization of HDAC6. HCT116 cells were deprived of valine, leucine, or isoleucine for 6 h followed by immunofluorescence analysis of cellular localization of HDAC6. Scale bar, 10 μm. d, Cell fractionation analysis was performed using HCT116 cells deprived of amino acids for 6 h, and then add an indicated amino acid (200 mM) for another 6 h. e, The half-maximal concentration of valine at the cellular level to restrain HDAC6 in cytosol of Fig. 2d. f, g, Absolute quantification of valine in HCT116 cells upon valine deprivation for different times (f) or valine restriction at different concentration (g). h, The localization of HDAC6 was examined via cell fractionation assay after treatment with concentration gradient of SLC7A5 inhibitor JPH203 (0, 2, 4, 6, 8, 10 μM) for 24 h and then rescued with N-Methyl-L-valine (CH3-Val, 0.8 mM) for 6 h. i, Effects of BCAA transporter SLC7A5 on the subcellular localization of HDAC6. Cell fractionation assay was performed with SLC7A5 wildtype or knockdown HCT116 cells upon valine deprivation for 6 h and then rescued with L-valine-OMe (Val-Ome, 0.8 mM) for 6 h. j, Effects of valine and its analogues on cellular localization of HDAC6 under the condition of valine deprivation. Cell fractionation analysis was performed in HCT116 cells deprived of valine for 6 hrs before valine or one of its analogues (10 mM) were added for 6 hrs. k, Effects of valine deprivation on the location of Flag-HDAC6 and its various truncations. Cell fractionation analysis was performed with HCT116 cells transfected with Flag-HDAC6 and its various truncations. l, Quantification of HDAC6 nuclear and cytoplasmic localization for Fig. 2f. For e–g,l, data are presented as mean ± s.d. (n = 3 independent experiments). Statistical analysis was performed using one-way ANOVA (f, g, l); ****P < 0.0001, NS, not significant. For gel source data, see Supplementary Fig. 1.
