Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot of histone Kla levels on nucleosomes isolated from HEK-293T cells that were incubated with recombinant Sirt6 and/or NAD + . (B) Quantitation of the blot from panel A. Densitometry data were corrected based on the total protein stain then normalized to the untreated control condition. Groups were compared using a one-way ANOVA followed by Tukey’s post hoc test. n=3, error plotted as S.D. p (-/-v. +/+) = 0.006. (C) Western blot analysis of time courses of enzymatic deacylation reactions using 25 nM substrate nucleosome, 100 nM Sirt6, and 1 mM NAD + . (D) Quantitation of western blot data for the H3K9Ac substrate in panel C. V I was calculated for each substrate concentration by fitting the first three data points from this curve using standard linear regression. (E) As in panel D, but for the H3K9La substrate. (F) V I values plotted as a function of substrate concentration for each PTM substrate. For panels D, E, and F, n=3, error plotted as S.D.

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, Isolation, Incubation, Recombinant, Quantitation Assay, Staining, Control, Concentration Assay

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

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 (PTM BIO) to analyze Kla levels on acid-extracted histones from wild type and Sirt6 knockout U2OS cells in the presence of a titration of rotenone, a mitochondrial Complex I inhibitor. Total protein was measured using a fluorescent total protein stain (LI-COR). (B) Antibody signal was corrected based on the loading control signal, then normalized 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=0.0005 (C) As in panel A but using the pan-acetyllysine antibody to measure levels of histone Kac. (D) Quantitation of data from panel C. Data were processed as in panel B. n=3, error plotted as S.D.

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, Quantitation Assay

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Levels of various histone acyl PTMs on acid-extracted histones from wild type or Sirt6 knockout U2OS cells were measured using site-specific antibodies (CST and PTM BIO). Total protein in each sample was measured using a fluorescent total protein stain (LI-COR). (B) Quantitation of data from panel A. Data were corrected based on the Revert 700 total protein stain, then normalized to the - Lactate control condition. Statistical analysis was performed using Student’s t-test and corrected for multiple hypothesis testing using the Holm-Šídák correction. n=3, error plotted as S.D. H3K9La p=0.013, H3K18La p=0.019.

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: Knock-Out, Staining, Quantitation Assay, Control

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot measuring histone Kla on acid-extracted histones in WT and Sirt6-KO U2OS cell lines not treated with supplemental L-lactate with and without overexpression of human Sir6 under the control of a CMV promoter. WT histone H3 (CST) is used as a loading control. (B) Western blot measuring Sirt6 in the soluble protein fraction from cell lines from (a). β-Actin is used as a loading control. (C) Kla signal from (a) was quantified, normalized to the loading control, and represented as a fold change from the average of the WT condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (D) as in (a) but measuring histone Kac. (E) as in (c), but for Kac data from (d) (F) As in (a), but the cells were treated with 25 mM sodium L-lactate for 24 hours prior to histone extraction. (G) As in (b), but the cells were treated with 25 mM sodium L-lactate for 24 hours prior to cell lysis. (H) Kla signal from (f) was quantified, normalized to the loading control, and represented as a fold change from the average of the WT condition. (I) Inverse correlation of data from (f) and (g). Sirt6 signal was normalized by dividing by the β-actin loading control. Kla signal was normalized as in (h). The data were analyzed using a standard linear regression, R 2 = 0.84.

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, Over Expression, Control, Extraction, Lysis

Journal: bioRxiv

Article Title: Sirtuin 6 is a histone delactylase

doi: 10.1101/2024.09.28.615627

Figure Lengend Snippet: (A) Western blot measuring histone Kla in WT and Sirt6-KO U2OS cells treated with sodium L-lactate and panobinostat, as indicated. A total protein stain (LI-COR) was used as a loading control. (B) As in panel A but measuring histone Kac. (C) Quantitation of selected data from panel A. Histone Kla signal was quantified using densitometry and corrected based on the total protein stain. Data are presented as a fold change from the untreated (-lactate, -panobinostat) condition. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. p 1 = 0.03, p 2 = 0.02, p 3 =0.005, p 4 =0.002. n=3, error plotted as s.d. (D) Quantitation of selected data from panel B. Data was processed as in panel C. n=3, error plotted as s.d. Quantitation of additional conditions is available in figure S4.

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, Staining, Control, Quantitation Assay

Journal: bioRxiv

Article Title: A high-density lineage tree reveals dynamics of expression differences accumulation in nondifferentiating clonal expansion

doi: 10.1101/2021.11.24.469964

Figure Lengend Snippet: ( A ) RT-PCR results for the RNA expression levels of the STADT barcode and Cas9 in cells growing in the presence of different concentrations of cumate. GAPDH was used as an internal control. The STADT barcode and Cas9 were amplified for 30 cycles, while GAPDH was amplified for 26 cycles. ( B ) RT-qPCR results for the expression levels of the STADT barcode relative to those of GAPDH. ( C ) RT-qPCR results for the expression levels of the Cas9 gene relative to those of GAPDH in cells cultured with different concentrations of cumate. ( D ) Western blot of the expression levels of Cas9 in cells cultured with different concentrations of cumate. GAPDH was used as an internal control. The small bands (*) may have resulted from the degradation of the Cas9 protein. ( E ) RT-PCR results showing the expression levels of sgRNA1 in the cells infected with different MOIs (0.1, 30) of the sgRNA lentivirus. U6 was used as an internal control. sgRNA1 was amplified for 35 cycles, while U6 was amplified for 30 cycles. ( F ) RT-qPCR result showing the expression level of sgRNA1 relative to that of U6 in the cell lines infected with different MOIs (0.1, 30) of the sgRNA lentivirus.

Article Snippet: For the sgRNAs, two DNA cassettes, U6>sgRNA1-U6>sgRNA2 and U6>sgRNA3-U6>sgRNA4, were synthesized and subsequently combined by Golden Gate ligation in the sgRNA vector pLV-U6>sgRNA1-U6>sgRNA2-U6>sgRNA3-U6>sgRNA4 (VectorBuilder, no. VB180515-1178wxn).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Amplification, Quantitative RT-PCR, Expressing, Cell Culture, Western Blot, Infection