Journal: Scientific Reports

Article Title: ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation

doi: 10.1038/s41598-022-24184-8

Figure Lengend Snippet: Altered doses of ABE8e and sgRNA treatment illustrate a dose-dependent relationship for bystander editing and protein restoration. ( a ) ABE8e and sgRNA were administered at high, medium and low doses and Sanger sequencing analysis performed post-editing to assess outcomes at mutation locus (c.5047) and the nucleotide in position 3 affected by bystander editing (c.5052). High dose = 5 µg sgRNA + 2 µg ABE8e, Med. dose = 1 µg sgRNA + 2 µg ABE8e, Low dose = 0.5 µg sgRNA + 1 µg ABE8e, ABE7.10 = 1 µg sgRNA + 2 µg ABE7.10. ( b ) NGS analysis of treatment outcomes at the c.5047 mutation locus found decreasing the High dose to Medium led to only a slight decrease in editing correction from 94.2 to 91.6%. The low dose engendered a correction of 45.9% of mutated alleles which was higher than ABE7.10 with an efficiency of 10.8%. Statistical difference and error bars were calculated using the Wald method. ( c ) NGS analysis of bystander editing at the c.5052 locus found that decreasing the ABE8e dose from High to Medium led to a large decrease in bystander editing, from 75.5 to 49.2%. Decreasing the ABE8e dose further resulted in 20.1% conversion of the bystander base, with ABE7.10 treatment causing 12.2% bystander editing. Statistical difference and error bars were calculated using the Wald method. ( d ) Western blots of protein collected from lysate of cells untreated and treated with different doses of ABE8e detected differential concentrations of a 290 kDa band, corresponding to full-length C7, using a polyclonal C7 antibody. Densitometric analysis of Western blots normalised to WT expression found EB cells treated by the highest dose of ABE8e expressed significantly more C7 in the cell lysate compared to untreated EB fibroblasts. There was no significance difference in C7 expression between the WT and the ABE8e High dose conditions nor between any of the untreated EB fibroblasts and the ABE8e Medium dose, Low dose and ABE7.10 conditions. N = 4 for WT, EB and ABE8e High dose conditions. N = 3 for ABE8e Medium dose and N = 2 for ABE8e low dose and ABE7.10 conditions. Representative blot shown. Significance found using the T -test. Cropped blots shown, full blots displayed in Supplementary Fig. S5a,b,e Western blots of cell medium collected from untreated and treated cells and probed by the polyclonal C7 antibody detected a 290 kDa band corresponding to secreted full-length C7. Densitometric analysis normalised to WT expression levels found EB cells treated by the highest and medium doses of ABE8e secreted similar C7 levels to Wild Type fibroblasts (WT) and more than untreated EB cells. EB fibroblasts treated with the low dose of ABE8e engendered a marginal increase in C7 whereas expression after ABE7.10 treatment was largely unchanged. n = 1. Full blots of C7 and Ponceau staining displayed in Supplementary Figs. S5c and S5d.

Article Snippet: The membrane was then incubated with a rabbit anti-collagen VII polyclonal primary antibody (Bio-Rad, VPA00854) diluted 1:1000 in 5% Bovine Serum Albumin (BSA) in TBS-1% Tween (TBS-T, Bio-Rad) overnight at 4 °C.

Techniques: Sequencing, Mutagenesis, Western Blot, Expressing, Staining

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: NOX4 regulates cell–cell adhesion in HCC cells. ( a ) NOX4 expression levels by quantitative PCR. Data represent the mean±s.e.m. ( N =4). ( b ) NOX4 and E-cadherin protein levels by western blot. β-actin was used as loading control. A representative experiment is shown ( N =3). Quantification normalized by loading control is shown under each blot. ( c ) Immunofluorescence of E-cadherin (green) and DAPI (blue; scale bar, 50 μm). ( d ) Representative bright-field images of cells cultured at basal conditions on top of plastic (left). Cell–cell contact imaging by immunofluorescence of E-cadherin (green) and ZO-1 (green); DAPI (blue; N =3; scale bar, 50 μm; right). ( e ) Analysis of CDH1 (E-cadherin) expression in PLC/PRF/5 cells by quantitative PCR. Data are mean±s.e.m. ( N =4). ( f ) E-cadherin protein levels by western blot. β-actin was used as loading control. A representative experiment is shown ( N =3). ( g ) Representative bright-field images of human HCC cells cultured on top of a bovine collagen I matrix (scale bar, 50 μm). ( h ) Quantification of the percentage of individual cells in each case. ( i ) Representative bright-field images of cells on top of a bovine collagen I matrix when NOX4 expression is silenced (scale bar, 50 μm). ( j ) Quantification of the percentage of individual cells in each case. ( k ) Number of cells per group. In ( h , j and k ) data are mean±s.e.m. ( N =4). See also .

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Cell Culture, Imaging

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: NOX4 suppresses the migratory/invasive potential of HCC cells. ( a ) Manual tracking of NOX4-silenced cells moving on a thick layer of collagen I/matrigel. Quantification of speed ( b ), distance ( c ) and directionality ( d ) of individual migrating cells. Data are expressed as mean±s.e.m. (20–30cells per condition). See also . Quantification of 3D invasion into a matrix of bovine collagen I (left) and collagen I/matrigel mix (1:1; right) of different human HCC cell lines ( e ) and of NOX4 knocked down Huh7 and PLC/PRF/5 cells ( f ). Data are expressed as fold increase versus Huh7 cells or shControl and are mean±s.e.m. ( N =3–5). ( g ) Invasive growth analysed embedding cells as spheroids in a bovine collagen I matrix. Representative images at day 0 and 4 (scale bar, 100 μm). ( h ) Representative images and quantification of the increase in area of the spheroids from control (shControl) and NOX4-silenced (shNOX4) cells between day 0 and 4 after embedding them into collagen (scale bar, 100 μm). ( i ) Percentage of HCC patients showing NOX4 deletion (23%) from the The Cancer Genome Atlas database ( N =249). Proportion of HCC patients in tumour grade 1/2 (17% left) and tumour grade 3/4 (32% right) showing NOX4 deletion.

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques:

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: NOX4 suppresses actomyosin contractility in HCC cells. In all experiments cells were cultured on top of a bovine collagen I matrix. ( a and d ) Percentage of cells with blebs. ( b and e ) quantification of phosphorylated MLC2 (pMLC2) immunostaining intensity per cell surface. ( c and f ) Representative confocal images of pMLC2 (green), F-actin (red) and DAPI (blue) staining ( N =4; scale bar, 20 μm, square with a higher magnification is shown for better visualisation). White arrows indicate blebs. Data in ( a , b , d and e ) are mean±s.e.m. ( N =4, at least five pictures per experiment). ( g ) Phosphorylated and total MLC2 protein levels. GAPDH was used as loading control. A representative experiment is shown ( N =3). ( h ) 3D imaging of PLC/PRF/5 (shControl and shNOX4) and SNU449 cells invading a collagen I/matrigel mix (1:1) matrix. Higher magnification is shown for better visualization. Black arrow indicates direction of invasion.

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques: Cell Culture, Immunostaining, Staining, Imaging

Journal: Oncogene

Article Title: The NADPH oxidase NOX4 represses epithelial to amoeboid transition and efficient tumour dissemination

doi: 10.1038/onc.2016.454

Figure Lengend Snippet: Overexpression of NOX4 in SNU449 cells switches to a parenchymal phenotype, and suppresses actomyosin contractility and invasive behaviour. ( a ) NOX4 mRNA (up) and protein (down) levels in SNU449 cells overexpressing NOX4 (+NOX4) and in Huh7 cells. Beta-actin used as a loading control. ( b ) ZO-1 (green) and Vinculin (green) immunofluorescence. DAPI (blue) to detect nuclei ( N =3; scale bar, 50 μm). ( c ) Representative confocal images of immunostaining of pMLC2 and F-actin of HCC cells cultured on top of bovine collagen I matrix ( N =3; scale bar, 50 μm). ( d ) Phosphorylated and total MLC2 protein levels. GAPDH was used as loading control. A representative experiment is shown ( N =3). ( e ) Quantification of pMLC2 immunostaining intensity per cell surface. ( f ) Quantification of the percentage of individual cells cultured on top of a collagen I matrix. ( g ) Manual tracking of SNU449 cells moving on a thick layer of collagen I/matrigel. Quantification of speed ( h ) and distance ( i ) of individual migrating cells. Data are mean±s.e.m. (20–30cells per condition). ( j ) Quantification of 3D invasion into a bovine collagen I matrix. ( k ) Representative images of spheroids from control and NOX4 overexpressing cells at day 0 and 2 after embedding them into collagen I (scale bar, 100 μm). ( l ) Quantification of the increase in area of the spheroids between day 0 and 2. Data in ( a , c , f , j and l) are mean±s.e.m. ( N =3).

Article Snippet: For 3D invasion assays, cells were resuspended in serum-free bovine collagen I solution at 2.3 mg/ml or in a solution of serum-free bovine collagen I/matrigel (Corning, New York, NY, USA) in a 1:1 proportion, to a final concentration of 14 000 cells per 100 μl of matrix and spun down, in a 96-well plate.

Techniques: Over Expression, Immunofluorescence, Immunostaining, Cell Culture