Figure 3. cWFW reduces membrane fluidity in vivo and in vitro.

(a) The fluidity of the cytoplasmic membrane was measured for B. subtilis cells upon 10 min incubation with increasing concentrations of cWFW using the fluidity-sensitive fluorescent dye laurdan. Please note that high laurdan generalised polarisation (GP) correlates with low membrane fluidity. The diagram depicts the average and standard deviation of three replicate measurements. (b) Time-resolved laurdan generalised polarisation (GP) was measured upon addition of 6 μM cWFW. As a positive control, 50 mM of the membrane fluidiser benzyl alcohol (BA) was added to a replicate sample. The time point of addition is indicated with a dashed line. The graph depicts a representative measurement of three independent replicates. (c) Laurdan GP was measured for large unilamellar vesicles (LUVs) formed of E. coli polar lipid extract in the presence of increasing concentrations of cWFW. The peptide-to-lipid molar ratios (P/L) are indicated below the graph. See supplementary Figure S3b for examples of the recorded spectra. (d) Laurdan GP in the presence and absence of cWFW (P/L = 0.02) was measured for LUVs with varying lipid compositions (lipid molar ratios: PE/CL 87.5/12.5, PE/PG 75/25). See supplementary Figure 3c–f for recorded spectra. PE: palmitoyl-oleoyl-phosphatidylethanolamine, PG: palmitoyl-oleoyl-phosphatidylglycerol, CL: cardiolipin and E. coli: E. coli polar lipid extract. (e) Average and standard deviation of cWFW-induced changes in laurdan GP (ΔGP) are shown for the different lipid compositions and peptide-to-lipid molar ratios (P/L) from two independent experiments. The statistical significances were calculated using unpaired (panels a) and paired (panel e) two-tailed Student t test.