Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Analysis of Nucleoporin Function Using Inducible Degron Techniques

doi: 10.1007/978-1-0716-2337-4_9

Figure Lengend Snippet: Design of gRNAs for targeting the NUP153 gene. (a) Position and sequences of gRNAs targeting last exon of NUP153 gene. SpCas9 cut sites on genomic DNA are indicated with blue arrows, gRNA sequences are highlighted in orange, PAM sequences are shown in yellow. Note, PAM sequence should be present only in genomic DNA. (b) Cloning of gRNA sequences with added CACC and CAAA overhangs into pX330 vector. gRNA-1 requires additional G nucleotide in the beginning of gRNA sequence to initiate expression of gRNA from U6 promoter. Note that the gRNA-9 starts from a G nucleotide and does not require additional G nucleotide

Article Snippet: 3.1. gRNA Cloning We used the pX330 vector (Addgene, 42230) to drive simultaneous expression of S. pyogenes Cas9 (SpCas9) protein and guide RNA (gRNA) sequence in the same cell.

Techniques: Sequencing, Cloning, Plasmid Preparation, Expressing

Journal: Nucleic Acids Research

Article Title: SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity

doi: 10.1093/nar/gkz648

Figure Lengend Snippet: ( A ) qPCR analysis of relative mRNA expression levels of mTOR signalling genes in WT and SIRT6-KO mice heart tissues. n = 4–10 mice per group. Data are presented as mean ± s.d, * P < 0.05. AKT1 was used as a positive control. GAPDH was used as a negative control. ( B ) ChIP analysis to detect SIRT6 binding to the promoters of the indicated genes performed with a SIRT6-specific antibody or IgG control antibody in WT 293T cells. n = 3–4. Data are presented as mean ± s.d, * P < 0.05. ( C ) ChIP analysis to detect Sp1 binding to the promoters of the indicated genes performed with a Sp1-specific antibody or IgG control antibody in WT 293T cells. n = 3–4. Data are presented as mean ± s.d, * P < 0.05. ( D ) Representative images of western blotting analysis of mTOR signalling under Sp1 depleted (Sp1-KD) conditions in 293T cells. The Sp1 levels were depleted by transfecting the cells with specific shRNA and the knockdown was confirmed by western blotting. (E) Representative images of western blotting analysis of mTOR signalling in Sp1-KO HeLa cells. Sp1 was deleted using a specific guide RNA and the CRISPR-Cas9 system. ( F ) Representative images of western blotting analysis of mTOR signalling under Sp1 overexpression conditions in 293T cells. The cells were transfected with empty vector or Sp1 expressing plasmid for 48 h and Sp1 overexpression was confirmed by immunoblotting. ( G ) Sp1 was immunoprecipitated from 293T cells and the interaction with SIRT6 was tested by western blotting. IgG was used as control. Whole cell lysate (WCL) was probed for indicated proteins by western blotting. The results presented here are representative of three independent experiments. ( H ) Luciferase reporter assay to assess the transcriptional activity of Sp1 was performed in pcDNA, SIRT6-WT or SIRT6 H133Y expressing 293T cells. The results are expressed as the fold change relative to pcDNA expressing cells. n = 6. Data are presented as mean ± s.d, * P < 0.05. ( I ) Realtime qPCR analysis of relative mRNA levels of GLUT1 and GLUT3 in WT and SIRT6-KO mice heart tissues. n = 6 mice per group. Data are presented as mean ± s.d, * P < 0.05. ( J ) Sp1 was immunoprecipitated from the 293T cells transfected with pcDNA, SIRT6-WT or SIRT6-H133Y plasmids and western blotting analysis was performed to detect the levels of Sp1 acetylation (Ac-Lys) by anti-acetyl-lysine antibody. IgG was used as a negative control in this assay. Whole cell lysate (WCL) was probed for indicated proteins by western blotting. (K) Representative immunofluorescence images depicting the levels and localization of Sp1 transcription factor (green) under control and SIRT6-KD conditions in Hela cells. SIRT6 was depleted using specific siRNA and knockdown was confirmed by immunostaining for SIRT6 (green) in a parallel experiment . The nuclei were stained with Hoechst 33342 and are shown in blue. Scale bar = 20 μm.

Article Snippet: For generation of stable Sp1 knockout cell line, gRNA targeting Sp1 cloned in plentiCRISPR v2 vector was obtained from GenScript ® .

Techniques: Expressing, Positive Control, Negative Control, Binding Assay, Control, Western Blot, shRNA, Knockdown, CRISPR, Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Immunofluorescence, Immunostaining, Staining

Journal: Nucleic Acids Research

Article Title: SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity

doi: 10.1093/nar/gkz648

Figure Lengend Snippet: ( A ) ChIP analysis to detect changes in Sp1 binding to the Rheb, mTOR and p70S6K promoter regions in control or SIRT6 stable knockdown 293T cells performed with Sp1 or IgG control antibody. n = 3. Data are presented as mean ± s.d, * P < 0.05. ( B ) ChIP analysis to detect changes in acetylation status at H3K9 residue at Rheb, mTOR and p70S6K promoter regions in control or SIRT6 stable knockdown 293T cells performed with Sp1 or IgG control antibody. n = 3. Data are presented as mean ± s.d, * P < 0.05. ( C ) ChIP analysis to detect changes in acetylation status at H3K56 residue at Rheb, mTOR and p70S6K promoter regions in control or SIRT6 stable knockdown 293T cells performed with Sp1 or IgG control antibody. n = 3. Data are presented as mean ± s.d, * P < 0.05. ( D ) Overlay of 2D 15 N- 1 H TROSY-HSQC NMR spectra of free Sp1 ZFDBD (black) with Sp1 ZFDBD in complex with SIRT6-WT (in red) (at 1:2 molar ratio). The disappeared cross peaks upon addition of SIRT6 are highlighted in box. ( E ) Overlay of 15 N- 1 H TROSY-HSQC NMR spectra of free Sp1 ZFDBD (black) with Sp1 ZFDBD in complex with mutant SIRT6-H133Y (in red) (at 1:2 molar ratios). The disappeared cross peaks upon addition of mutant SIRT6-H133Y are highlighted in box. ( F ) Representative images of western blotting SUnSET analysis in control or SIRT6-KD HeLa cells in the presence vehicle or 200 nM of Sp1 inhibitor Mithramycin A. DMSO was used as a vehicle control. SIRT6 was depleted using specific siRNA and the knockdown was confirmed by immunoblotting for SIRT6. Reduction in mTOR phosphorylation was used to confirm the action of Mithramycin A. ( G ) Quantitative representation of cap-dependent translation rates measured using the luciferase reporter pRL-CMV in control or SIRT6 depleted HeLa cells (siRNA mediated knockdown) in the presence or absence of 200 nM Mithramycin A. Renilla luciferase readings were normalized against the mRNA levels of Renilla luciferase. n = 4. Data are presented as mean ± s.d, * P < 0.05. ( H ) Quantitative representation of cap-dependent translation rates measured using the luciferase reporter pRL-CMV in control or SIRT6 depleted HeLa cells (siRNA mediated knockdown) under control or Sp1 depleted conditions (shRNA mediated knockdown). Renilla luciferase readings were normalized against the mRNA levels of Renilla luciferase. n = 4. Data are presented as mean ± s.d, * P < 0.05.

Article Snippet: For generation of stable Sp1 knockout cell line, gRNA targeting Sp1 cloned in plentiCRISPR v2 vector was obtained from GenScript ® .

Techniques: Binding Assay, Control, Knockdown, Residue, Mutagenesis, Western Blot, Phospho-proteomics, Luciferase, shRNA