Visualization of the ECM, presence of HMWGP protein and FLO11 mRNA in BR-RF colonies. A. Glycoproteins in extracts from BR-F, BR-S and BR-RF colonies stained with silver (Ag) and visualized by the ConA/peroxidase (ConA) method; arrows indicate HMWGP in BR-F (black arrows) and in BR-RF (red arrows) colonies. B. Visualization of the ECM in vertical cross-sections of BR-RF-pGAL1-GFP colonies using 2P-CM. Green, GFP fluorescence indicates areas in which the inducer (galactose) reached the cells; red, autofluorescence of all colony cells visible in areas where the ECM prevented the inducer from accessing the cells. Intact colonies were induced from the bottom by placing them for 5 h on agar soaked with 2% galactose . C. FLO11 mRNA in 4- and 7-day-old BR-F, BR-RF and BR-S colonies. Right, stronger exposure revealed low FLO11 mRNA levels in BR-S colonies. The discontinuous line compares the length of the FLO11 transcripts in BR-RF and BR-S cells. The RDN18 gene was used as an RNA loading control.