Journal: bioRxiv
Article Title: Sirtuin 6 is a histone delactylase
doi: 10.1101/2024.09.28.615627
Figure Lengend Snippet: (A) Western blot using a pan-lactyllysine antibody to analyze Kla levels on acid-extracted histones from wild type (“WT”), Sirt6 knockout (“S6KO”), and Sirt7 knockout (“S7KO”) U2OS cells in the presence of a titration of sodium L-lactate. Total protein was measured using a fluorescent total protein stain. (B) As in panel A but using a pan-acetyllysine antibody to analyze levels of histone Kac. (C) Western blots from panel A quantified by normalizing Kla signal to total protein then represented as a fold-change from the untreated condition. n=3, error plotted as S.D. Slopes of linear regressions were compared using Welch’s t-test. p (slope, S6KO v. WT) < 0.0001. p (slope, S7KO v. WT) = 0.39. (D) Western blots from panel B quantified by normalizing Kac signal to total protein then represented as a fold-change from the untreated condition. For WT and S6KO, n=3. For S7KO, n=2. Error plotted as S.D. (E) Baseline histone lactylation in untreated cells was measured using a pan-Kla antibody as in panel A and normalized to the loading control. The blot images used to generate this plot are shown in . Error plotted as S.D., p = 0.68.
Article Snippet: For mammalian cell overexpression, the full length, human Sirt6 sequence with a C-terminal FLAG tag was cloned into a pLJM1 mammalian expression vector with a CMV promoter (“pLJM1-Sirt6-3xFLAG”)., For the human cell knockouts, the following sgRNA sequences were cloned into the pSpCas9(BB)-2A-Puro (PX459) V2.0 vector: Sirt6: sgRNA1-CCTGAAGTCGGGGATGCCAG, sgRNA2-TACGTCCGAGACACAGTCGT Sirt7: sgRNA1-CGTTACCAGGTCCGCGCTCT, sgRNA2-GCTTCAGGCCCTCGCGCCGC, sgRNA3-GGCCCTGCAGCTCCGTTACC The oligonucleotide design and cloning were performed according to the protocol provided by the Zhang lab on the Addgene #62988 website.
Techniques: Western Blot, Knock-Out, Titration, Staining, Control
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