atthophos alkaline phosphatase (ap) fluorescent substrate system  (Promega)

Journal: bioRxiv

Article Title: Mechano-dependent sorbitol accumulation supports biomolecular condensate

doi: 10.1101/2023.07.24.550444

Figure Lengend Snippet: (a) Representative images of primary mammary tumors are shown as isolated from a cohort of 100 patients. Picrosirius red staining visualized by both parallel and orthogonal light displays tumor ECM remodeling, and representative IHC images show Ki67, α-SMA, and TAZ staining. Scale bar=100 µm. (b-c) Correlations between TAZ staining and ECM remodeling (b) and between TAZ staining and proliferation (c) are shown. Pearson correlation coefficients (R ) are indicated. (d) Representative immunofluorescence images and quantification of 2-NBD-Glucose intensity of MDA-MB-231 cells cultivated in 3D after the indicated treatment. Scale bar=100 µm. (e) Quantification of 13 C-glucose incorporation in sorbitol in MDA-MB-231 cells cultivated in 3D after the indicated treatment. (f-g) Representative immunofluorescence images and quantification of cell with GFP-TAZ condensates and Ki67 of MDA-MB-231 cells cultivated in 3D after the indicated treatment. Scale bar: Upper panel=100 µm and lower panel=20 µm. (h) ChIP-qPCR data from cells treated as indicated at CTGF, ANKRD1 and CR61 locus. Results are expressed as percentage of total input DNA prior to immunoprecipitation with anti-TAZ or anti-IgG control. In all the panels n>12 spheroids from 3 independent experiments were analyzed. ns=not significant; **P<0.01; ***P<0.001; (d-h) Bonferroni’s multiple comparison test; data are mean ± s.e.m of at least n=3 independent experiments.

Article Snippet: Cells were incubated with 20uM of 2-NBD-Glucose Fluorescent glucose uptake probe (Abcam, ab146200) in glucose-free medium for 20min at 37°C.

Techniques: Isolation, Staining, Immunofluorescence, Immunoprecipitation