Journal: The Journal of pathology

Article Title: A 3D tri-culture system reveals that activin receptor-like kinase 5 and connective tissue growth factor drive human glomerulosclerosis

doi: 10.1002/path.4960

Figure Lengend Snippet: (A) MC 3D monoculture without the addition of TGFβ, and (B) in the presence of TGFβ, shown by H&E staining of paraffin-embedded sections. Human collagen type I/III (immunostained with an antibody that recognises both collagen type I and III) (C) and human collagen type IV (D) can be demonstrated in nodules by immunoperoxidase staining. (E) High-power view of MC nodule by scanning electron microscopy in cross section. (F) MC nodule count and collagen type I alpha1 (COL1a1), and collagen type IV alpha1 (COL4a1) RNA quantification (n=3). (G) Effect of ALK5 inhibition (Alk5i) on MC nodule formation (n=3). (H) Effect of SMAD2 or SMAD3 siRNA knockdown in MCs on nodule formation (n=3) with immunoblots demonstrating degree of knockdown. [DharmaFECT (DH), non-targeting siRNA control (siCP), siSMAD2 (siS2), siSMAD3 (siS3), total SMAD2 (tSMAD2), total SMAD3 (tSMAD3)]. Error bars represent 1 s.d. NS, not significant; ***, p<0.001; **, p<0.01: 2-tailed Student’s t test.

Article Snippet: After culture, matrices were embedded in HistoGel, fixed in 4% paraformaldehyde and paraffin wax-embedded for sectioning and staining for human collagen type IV (mouse anti-human collagen IV monoclonal antibody 2150–0121, Bio-Rad, CA, USA) and human collagen type I/III (rabbit anti-human collagen I/III polyclonal antibody 2150–2210, Bio-Rad, CA, USA).

Techniques: Staining, Immunoperoxidase Staining, Electron Microscopy, Inhibition, Western Blot

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