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: Foods

Article Title: Extracellular Heme Proteins Influence Bovine Myosatellite Cell Proliferation and the Color of Cell-Based Meat

doi: 10.3390/foods8100521

Figure Lengend Snippet: Two-dimensional immunofluorescence stain of isolated bovine muscle satellite cells (BSCs). ( A ) Proliferating bovine satellite stained for DAPI, actin cytoskeleton (Phalloidin), and Pax7, a nuclear marker of satellite cells. Stains show a highly pure satellite cell population, following isolation and pre-plating protocol. ( B ) Following one week of differentiation, cells were stained for DAPI, actin cytoskeleton (Phalloidin), and Troponin T (CT3), a marker of myogenesis. Scale bars are 200 µm.

Article Snippet: For BAMs, secondary antibodies for Troponin T were diluted in blocking solution containing DAPI (Thermo Fisher, #62248, 1:1000) and added to constructs for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Isolation, Marker

Journal: Foods

Article Title: Extracellular Heme Proteins Influence Bovine Myosatellite Cell Proliferation and the Color of Cell-Based Meat

doi: 10.3390/foods8100521

Figure Lengend Snippet: Confocal immunofluorescent imaging of BAMs. BAMs generated from BSC, BSC + Hb, or BSC + Mb (3 mg/mL for both heme proteins) were stained after eight days of differentiation for DAPI, actin cytoskeleton (Phalloidin), and Troponin T (CT3), a marker of myogenesis. Images show multinucleated myotube formation in BSC and BSC + Mb constructs, though not in BSC + Hb constructs. Scale bars are 200 µm.

Article Snippet: For BAMs, secondary antibodies for Troponin T were diluted in blocking solution containing DAPI (Thermo Fisher, #62248, 1:1000) and added to constructs for 1 h at room temperature.

Techniques: Imaging, Generated, Staining, Marker, Construct