lentiviral particles harboring sirt6 overexpression vector  (Addgene inc)

Journal: Cell Discovery

Article Title: Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV

doi: 10.1038/s41421-021-00337-3

Figure Lengend Snippet: a RNA-sequencing was conducted with Huh7.5.1 cells transfected with Vector (Control) or prcccDNA and pCMV-Cre. Heatmap of 11 transcriptional DEGs, which have transcription corepressor activity, three biological replicates were shown for HBV and control group. b RT-qPCR confirmed that HBV downregulated the transcriptional level of endogenous SIRT6 transcription. c Network analysis of SIRT6-associated genes whose transcription was altered by HBV. Red and blue nodes indicate the upregulated and downregulated genes, respectively. The color intensity indicates the fold change level of the gene. Nodes with * are DEGs ( P -adjust <0.05, |log 2 FC| ≥ 1, also see methods). d RT-qPCR validation of some of the SIRT6-associated genes that were shown in c . e Representative profiles of ribosome footprints of human SIRT6 ORF upon HBV, translation initiation of endogenous SIRT6 was downregulated by HBV in Huh7.5.1 cells. The highest read count was set as 1 and the read counts of other groups were normalized to this group. f Endogenous SIRT6 was downregulated by HBV in Huh7.5.1 cells. g De novo infection of HBV downregulated endogenous SIRT6 level in HepG2-NTCP cells, HepG2 cells stably expressing NTCP (sodium taurocholate cotransporting polypeptide), the functional receptor of HBV. The results of two independent biological replicates were shown. h HBV downregulated SIRT6 in mouse livers infected with adenovirus harboring HBV genome. i Total proteins were extracted from the normal liver tissues of 12 patients who were diagnosed with HBV positive and negative, respectively. For patient information see Supplementary Table . Endogenous SIRT6 or GAPDH proteins were visualized with IB using anti-SIRT6 or anti-GAPDH, with their relative abundances calculated using ImageJ. j The reverse correlation between the serum levels of HBsAg and the relative abundances of endogenous SIRT6 protein in liver tissues of the 12 patients. * P < 0.05, ** P < 0.01, *** P < 0.001. qPCR results are presented as bar chart (For SIRT6, n = 2; for other genes, n = 3); ELISA data are presented as bar chart ( n = 3).

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: RNA Sequencing Assay, Transfection, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Infection, Stable Transfection, Expressing, Functional Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Discovery

Article Title: Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV

doi: 10.1038/s41421-021-00337-3

Figure Lengend Snippet: a , b Introduction of Sirt6 suppressed the expression of HBcAg ( a ) and HBsAg and HBeAg ( b ) antigens in Huh7.5.1 cells. c , d Knocking-down endogenous Sirt6 promoted the expression of HBcAg ( c ) and HBsAg and HBeAg ( d ) antigens in Huh7.5.1 cells. e , f Effect of SIRT6 enzymatic mutants on HBcAg ( e ) and HBsAg and HBeAg ( f ) expression in huh7.5.1 cells. g The effect of SIRT6 wild type or enzymatic deficient mutant on HBV genome replication was measured via southern blotting in HepG2 cells. h The chemical formula and a close-up view of MDL-800 in complex with 2′-O-acyl-ADP ribose (2′-O-acyl-ADPR) motif of human SIRT6 (X-ray structure, PDB number 5Y2F). i , j ELISA shows that MDL800 suppressed the expression of HBcAg ( i ) and HBsAg and HBeAg ( j ) antigens in dose-dependent manner in Tet-controlled HBV in HepAD38 cells. k MDL-800 treatment significantly suppressed HBV genome replication intermediates in Huh7 cells. RC relaxed circular, DNA DSL double strand linear DNA, SS single strand DNA. For ELISA, for panel b, n = 2; for others, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Expressing, Mutagenesis, Southern Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Discovery

Article Title: Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV

doi: 10.1038/s41421-021-00337-3

Figure Lengend Snippet: a SIRT6 co-immunoprecipitated with HBcAg in 293FT cells. b SIRT6 and HBcAg co-localized in the nuclei of Huh7.5.1 cells during HBV replication. Scale bars, 20 μm. c – f ChIP assays were performed using indicated antibodies with cells upon either HBV transfection alone ( c ), or in combination with SIRT6 overexpression ( d ) or knockdown ( e ) or MDL800 treatment ( f ). * P < 0.05, ** P < 0.01, *** P < 0.001. ChIP data were acquired in Huh7.5.1 cells and were presented as bar chart ( n = 3 per group).

Article Snippet: Lentiviral particles harboring SIRT6 overexpression vector (pCDH; Addgene) or sirt6 shRNA expression vector (pLKO.1; Sigma-Aldrich) were produced by transfection of HEK293FT cells with indicated plasmids and lentiviral packaging plasmid mix.

Techniques: Immunoprecipitation, Transfection, Over Expression