dapi-containing blocking solution p36966  (Thermo Fisher)

Journal: Life Science Alliance

Article Title: Novel determinants of NOTCH1 trafficking and signaling in breast epithelial cells

doi: 10.26508/lsa.202403122

Figure Lengend Snippet: (A) The primary screen was performed on MCF10A cells in a 384-well format. Genes belonging to a subset of the human genome were knocked down in six replicate plates for 72 h by RNAi along with the listed controls. In a duplicate experiment, cells were stimulated with EGTA. Control wells were distributed on the plates as shown. NT: non-targeting KD – cells were transfected with non-targeting siRNA. No siRNA: cells received RNAiMAX only. Positive control (pos ctrl) – cells were transfected with siRNA against PSENEN. Transfection pos ctrl: cells were transfected with siRNA against polo-like kinase 1 (PLK1) as a cytotoxic indicator of transfection efficiency. Outer wells (yellow) were filled with media to prevent evaporation in experimental wells. (B) To assess endogenous NOTCH1 localization, cells in plates were subjected to automated IF and DAPI/phalloidin labeling to demarcate nuclei and the cell cortex, respectively. The script steps of the image acquisition and analysis developed in-house are as follows: (1) The nuclei were segmented based on DAPI signal. (2) The cell surface was segmented using the phalloidin signal and overlaid on the NOTCH1 channel (3) to quantify levels of cell surface NOTCH1 (4), and the area between the cell cortex and the nuclei was used to determine levels of cytoplasmic NOTCH1 (5). (6) The phalloidin mask was also used to count cells and establish cell-to-cell boundaries. (7) Nuclear size was used as readout of cell viability as compact; pyknotic nuclei identify dead cells. Using such a pipeline, each gene KD was defined by its effects on the amount of NOTCH1 in the cell cortex, in the cytoplasm or in the nucleus.

Article Snippet: Next, the cells were incubated for 1 h with 1% BSA blocking solution containing DAPI at 1:4,500 (Cat #: D9542; Merck Life Sciences), phalloidin at 1:350 (Cat #: P5282; Merck Life Sciences), and Alexa Fluor 488 anti-rat secondary antibody at 1:400 (Cat #: A21208; Thermo Fisher Scientific).

Techniques: Control, Transfection, Positive Control, Evaporation, Labeling

Journal: Emerging Microbes & Infections

Article Title: Treponema pallidum protein Tp0136 promotes angiogenesis to facilitate the dissemination of Treponema pallidum

doi: 10.1080/22221751.2024.2382236

Figure Lengend Snippet: Tp0136 promoted angiogenesis in vivo and in vitro . (a) Effect of Tp0136 protein and antibody on blood vessels in lesions. Red arrows indicate the blood vessels. The bar graph indicates the number of the blood vessels. Scale bars = 100 mm. (b) Effect of Tp0136 on cell migration using transwell migration assay. The bar graph indicates the crystal violet area. Scale bars = 100 mm. The experiment was repeated three times independently for each group. (c) Effect of Tp0136 on spheroid-based sprouting. The bar graph indicates the number of sprouting. Scale bars = 100 mm. The experiment was repeated three times independently for each group. (d) Effect of Tp0136 on tubule formation. Tp0136 (10 mg/mL) was coincubated with HMEC-1 cells for 6 h, after which tubule formation was stained using Calcein AM. Bar graphs indicate the number of nodes, the number of meshes, and the total branching length. Scale bars = 500 mm. The experiment was repeated three times independently for each group. (e-f) Three-dimensional microfluidic angiogenesis system of angiogenesis processed by Tp0136. (g) Effect of Tp0136 on three-dimensional angiogenesis. The bar graph indicates the number and the average length of angiogenic sprouts. Phalloidin-iFluor 555-red, DAPI-blue. Scale bars = 100 mm. The experiment was repeated three times independently for each group. Data are expressed as the mean ± SD from three independent experiments. Student's t test was used to compare data between two groups. One-way ANOVA was used to compare the mean values of three or more groups with one independent variable. * P vs. Control, *, P <0.05, **, P <0.01, ***, P <0.001.

Article Snippet: The cytoskeleton and nuclei were stained via immunofluorescence, using a Phalloidin-iFluor 555 reagent (Abcam, UK) and an anti-fluorescence quenching blocking solution containing DAPI (Beyotime, China), respectively.

Techniques: In Vivo, In Vitro, Migration, Transwell Migration Assay, Staining, Control

Journal: Emerging Microbes & Infections

Article Title: Treponema pallidum protein Tp0136 promotes angiogenesis to facilitate the dissemination of Treponema pallidum

doi: 10.1080/22221751.2024.2382236

Figure Lengend Snippet: Tp0136 promoted angiogenesis via PI3K/Akt leading to the increase in vascular permeability. (a) Effect of LY294002 on Tp0136-induced tubule formation. Scale bars = 500 mm. Bar graphs indicate the number of nodes, the number of meshes, and the total branching length in tubule formation. The experiment was repeated three times independently for each group. (b) Effect of LY294002 on Tp0136-induced angiogenesis was analyzed using three-dimensional angiogenesis analysis. The bar graph shows the number and the average length of angiogenic sprouts. Phalloidin-iFluor 555-red, DAPI-blue. Scale bars = 100 mm. The experiment was repeated three times independently for each group. (c) Effect of LY294002 on Tp0136-induced vascular permeability in three-dimensional microfluidic angiogenesis system. Time is indicated in h, min, and s. Data are expressed as the mean ± SD from three independent experiments. One-way ANOVA was used to compare the mean values of three or more groups with one independent variable. * P vs. Control, ** P < 0.01, *** P < 0.001. # P vs. Tp0136, # P < 0.05, ## P < 0.01.

Article Snippet: The cytoskeleton and nuclei were stained via immunofluorescence, using a Phalloidin-iFluor 555 reagent (Abcam, UK) and an anti-fluorescence quenching blocking solution containing DAPI (Beyotime, China), respectively.

Techniques: Permeability, Control