Journal: Nature Communications

Article Title: Multi-pathway DNA-repair reporters reveal competition between end-joining, single-strand annealing and homologous recombination at Cas9-induced DNA double-strand breaks

doi: 10.1038/s41467-022-32743-w

Figure Lengend Snippet: a Cartoon depicting potential outcomes of a multi-pathway reporter cell-line designed to quantify DSB-repair by error-free c-NHEJ and HR. b Diagramatic representation of the genomic DSB-repair reporter construct DSB-Spectrum_V1. Expanded region shows the DNA sequence targeted by Cas9. The sequence of the BFP cDNA is displayed in blue, the PAM sequences of the sgRNA target sites are displayed in red. Arrows in the inset and scissors in the cartoon indicate the Cas9 cut sites. Ligation of the distal DSB ends by error-free c-NHEJ will remove the spacer sequence and restore the BFP gene. c, d DSB-Spectrum_V1 was integrated into the genome of HEK 293T cells by lentiviral infection, followed by expansion of a single-cell clone. The resulting DSB-Spectrum_V1 cell-line was transfected with Cas9 and an sgRNA targeting a control locus (AAVS1) or BFP, and analyzed by flow cytometry at 72 h after transfection. Panel c shows representative flow plots, and panel d shows the quantification of multiple experiments ( n = 4; mean ± SEM). e DSB-Spectrum_V1 cells were transfected with indicated siRNAs (NTsi = Non-Targeting control), followed by transfection with Cas9 and an sgRNA targeting a control locus or BFP. At 72 h after Cas9 transfection cells were analyzed by flow cytometry. Percentages of each fluorescent population in the BFPsg-transfected cells were corrected for the background percentages seen in the AAVS1sg-transfected cells. Depicted is the ratio of background-corrected percentages for each fluorescent population to that of the NTsi control ( n = 3; mean ± SEM; One-way ANOVA, post-hoc Dunnett’s). f Western blot of lysates from cells analyzed in panel e . Tubulin is used as loading control. g As in panel d , but including treatment with NU7441 (2 µM), and background-corrected as described in panel e ( n = 4; mean ± SEM; ratio paired t -test, two-tailed). Source data for panels d, e, and g are provided as a Source Data file.

Article Snippet: Membranes were blocked using either 5% skim milk or Blocking buffer for fluorescent WB (Rockland) in PBS, followed by antibody staining in Blocking buffer for fluorescent WB (Rockland) diluted 1:1 in PBS with 0.1% Tween-20.

Techniques: Construct, Sequencing, Ligation, Infection, Transfection, Control, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Scientific Reports

Article Title: Smoking induces increased apoptosis in osteoblasts: changes in bone matrix organic components

doi: 10.1038/s41598-023-33965-8

Figure Lengend Snippet: Percentage of Type V collagen in non-smokers, former smokers and smokers (n = 19, n = 22, n = 22 respectively). Significant differences were found in smokers compared to non-smokers (*p = < 0.047, Dunn's Test). Photomicrographs of collagen Type I in bone tissue from femoral head-neck transition area. Values were expressed as mean ± SE (×400 magnification).

Article Snippet: Slides were incubated overnight with rabbit polyclonal anti-collagen type I (1:100; Rockland) anti-collagen type V (1:100; Rockland), diluted in PBS solution.

Techniques: