Fig. 1. Retention of endogenous and heterologously expressed TSP1 in the ECM of cultured cells. (A) Analysis of ECM isolation conditions. Confluent 4-day cultures of COS-7 cells were fixed in 2% paraformaldehyde and permeabilised in 0.5% Triton X-100 (PFA), or extracted as indicated, then stained with FITC-phalloidin to visualise F-actin or DAPI to visualise DNA. (B) Production of endogenous ECM by C2C12 and COS-7 cells. Confluent 4-day cultures were extracted with 20 mM NH₄OH and stained as indicated for components of the insoluble ECM. (C) Biochemical analysis. NH₄OH-insoluble ECM from 4-day cultures of the indicated cells was solubilised in hot SDS-PAGE sample buffer containing 100 mM DTT, resolved on 10% polyacrylamide gels and analysed by immunoblotting for fibronectin (FN) (COS-7 and C2C12 cells) or TSP1 (control COS-7 cells and COS-7 transfected to express human TSP1). (D) COS-7 cells were transfected with human TSP1, cultured for 3 days and then fixed in 2% paraformaldehyde, permeabilised and stained for human TSP1 to visualise the intracellular pool, or extracted with 20 mM NH₄OH and the insoluble ECM stained for human TSP1. Right panel shows a single array of puncta; arrows indicate examples of individual puncta. Scale bars: 25 µm (A); 10 µm (B,D).