Fig. 3: Analysis of matrix components in LCA-induced biofilms.

A Representative maximum intensity projection of an LCA-induced biofilm stained with TOTO-1 (extracellular DNA and dead cells) and SYTO 60 (intact cells). Scale bars = 20 µm. B Crystal violet quantification of biofilm biomass for cultures that were grown in the presence of LCA only (“untreated”) or LCA + DNase I for 24 h. ***p < 0.001, Welch’s t test (n = 4). C Representative maximum intensity projection of an LCA-induced biofilm stained with SYPRO Ruby (general protein stain) and DAPI (DNA/cells). Scale bars = 20 μm. D Crystal violet quantification of biofilm biomass, as well as OD₆₀₀ measurement of total biomass (biofilm + planktonic), for cultures that were grown in the presence of LCA only (“untreated”) or LCA + proteinase K for 24 h. *** p < 0.001, Welch’s t test (n = 4). E Representative maximum intensity projection of an LCA-induced biofilm stained with Calcofluor White (cellulose) and FM1-43 (cell membrane). Scale bars = 20 µm. F Quantification of biofilm formation (crystal violet absorption at OD₅₅₀ normalized to OD₆₀₀ as a proxy for growth) for the WT vs. ΔbcsA (cellulose-null mutant) after 24 h of growth in the presence of 100 µM LCA. *** p < 0.001, Welch’s t test (n = 4).