Dmi6000 ffw

MSCs were washed and fixed with 40 g/L PFA for 30 min at RT. Cells were permeabilized with 0.1% (v/v) Triton X‐100 (Sigma-Aldrich) for 10 min, at RT, rinsed and blocked with 5 g/L BSA/ 0.1% (v/v) Triton X‐100 for 1 h at RT. Then, cells were incubated with primary antibody against collagen type I (COL1, 1:250, Rockland), osteopontin (OPN; 1:250, Santa Cruz Biotechnology), fibrillin-1 (FBN1; 1:100, Thermo Fisher Scientific), collagen Triple Helix Repeat Containing 1 (CTHRC1; 1:50, Santa Cruz Biotechnology) and vitronectin (VTN; 1:25, Santa Cruz Biotechnology) overnight at 4 °C. Cells were washed three times with PBS 1x, 5 min each, incubated with the respective secondary antibody (1:1 000; Thermo Fisher Scientific) for 1 h at RT and washed again. Cell nuclei were stained with 1 μg/mL DAPI (Invitrogen) for 5 min. The antibodies manufacturers and respective dilutions are detailed in Additional file 1: Table SIV. The stainings were visualized under the Leica DMi6000 FFW (Leica Microsystems). Cells incubated with the antibody diluent alone (without primary antibody), followed by incubation with the secondary antibody, were used as negative controls. Semi-quantification of the protein was performed using the ImageJ Fiji software, considering a minimum of 6 distinct fields per condition.

The biofilms were grown for 24 h on µ-Dishes 35 mm high, with a polymer coverslip bottom (ibidi GmbH, Gräfelfing, Germany), from a starting inoculum of 10⁶ CFU/mL in a tryptic soy broth (TSB; Liofilchem s.r.l., Roseto degli Abruzzi, Italy), for PA004 and Pa3, or in TSBG (TSB + 1% Glucose), for SA007 and SA011. Biofilms were equally formed either in the presence of the carboxypyrano-ant extract or carboxypyCy-3-glc, each at 64 µg/mL. After 24 h at 37 °C, the planktonic phases were removed and the biofilms were rinsed with phosphate buffered saline (PBS, Sigma-Aldrich/Merck Life Science S.L.U., Algés, Portugal) and then stained with the Live/Dead staining mixture (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo Fisher Scientific, Porto Salvo, Portugal) for 30 min at room temperature in the dark, rinsed again with the PBS, and then visualized under a fluorescence microscope Leica DMI6000 FFW (Leica Microsystems, Carnaxide, Portugal) or under a laser scanning confocal system Leica TCS SP5 II (Leica Microsystems, Carnaxide, Portugal), equipped with (i) an inverted microscope, Leica DMI6000-CS, using an HC PL APO CS 63× /1.30 glycerin 21 °C objective and the lasers diode 405 nm and DPSS561 561 nm; and (ii) the LAS AF software.

Biofilms of SA007 and KP010 MDR clinical isolates were allowed to grow on 35 mm high µ-Dishes with ibidi polymer coverslips (ibidi GmbH), in TSB and in TSBG (TSB + 1% Glucose), respectively. The test peptide constructs (PP4-3.1 and MeIm-PP4-3.1) were previously added to each respective medium at concentrations equal to at MIC, ½ × MIC and ¼ × MIC. In the control groups, no peptides were added. After 24 h, in the case of SA007 biofilms, or after 48 h in case of KP010 biofilms, at 37 ℃, they were stained using the Live/Dead staining mixture (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo Fisher Scientific, Waltham, MA, USA) as described by Coelho and co-workers [24 (link)], and then visualized under a fluorescence microscope (Leica DMI6000 FFW, Leica Microsystems, Carnaxide, Portugal).