Zen 3.2 blue edition software

For TMPRSS2 protein immunofluorescence, A549 and TT1 cells were either stimulated or not with IL1β for 24 hours. By contrast, for the evaluation of cell susceptibility to SARS-CoV-2 infection, cells were either stimulated or not with IL1β and the selected inhibitors for 8 hours as previously described. The infection was then performed, using Heat-inactivated SARS-CoV-2 (VR-1986HK, ATCC) at 4 TCID50/mL. 72 hours after the infection, cells were fixed with PFA 4% (Sigma) in PBS, blocked with blocking solution (PBS, 1% BSA, 0,02% NP40) and incubated with SARS-CoV-2 spike antibody (polyclonal, GeneTex) followed by anti-rabbit Alexa Fluor 488 antibody (polyclonal, Invitrogen). Nuclei were counterstained with Hoechst 33342 (Invitrogen) and actin was stained with Phalloidin TexasRed (Invitrogen). Fluorescence images were acquired using the Zeiss LSM 800 confocal laser scanning microscope. The fluorescent signal per cell was quantified using ImageJ software (Fiji) and the corrected total cell fluorescence (CTCF) was calculated. TMPRSS2 staining in A549 and TT1 cells was performed by using H-4 (Santa Cruz Biotechnology) antibody according to the indicated protocol. 3D reconstruction was performed using ZEN 3.2 Blue edition software (Carl Zeiss).

Cells were plated on glass coverslips to 70–80% confluence for 48 hr in growth media. Cells were fixed in 1 % formaldehyde diluted in PBS for 15 min. The cells were rinsed three times with PBS for 5 min and coverslips were blocked for 1 hr with 1 X PBS/ 5% goat serum/0.3% Triton X-100 and then incubated with E-cadherin antibody (#4A2) overnight. Cells were washed three times for 5 min with PBS and incubated in anti-mouse IgG Alexa Fluor 555 Conjugate (Cell signaling #4409) at a dilution of 1:500 for 1 hr. Coverslips were rinsed three times for 5 min in PBS and briefly rinsed in distilled water prior to mounting in Prolong Gold Antifade Reagent with DAPI (Cell signaling #8961). All Images were acquired using a Zeiss LSM-780 confocal microscope and processed using ZEISS ZEN 3.2 (blue edition) software.

Colonies of L. crispatus P17631 and L. paracasei I1688, cultivated overnight on blood agar plates, were used to prepare a bacterial suspension. Specifically, the colonies were suspended in 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) until they reached a turbidity of 2.5 ± 0.3 on the McFarland scale, equating to roughly 1 × 10⁸ CFU/ml. This suspension was then diluted at a ratio of 1:1000 and transferred into 1 ml of BHI within μ-Slide, 8-well glass bottom chamber slides (Ibidi, Germany). The bacterial mixture was incubated at 37 °C over 48 h for biofilm development. After this period, the medium was discarded, and the samples were rinsed with 0.45% saline solution. Biofilm cells were then stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), following the manufacturer's guidelines, and examined with an Apotome system (Zeiss, Oberkochen, Germany) connected to an Axio Observer inverted fluorescence microscope (Zeiss). Data were analyzed with the ZEN 3.2 (blue edition) software (Zeiss).

We seeded WI-38 and C2C12 cells at 1.0 × 10⁵ cells per well in a 35 mm MatTek microscopy glass dish (ThermoFisher Scientific, Waltham, MA, USA, cat. no. NC9574048) and treated with 20 μM of CCG-203971 and 20 μM of CCG-232601 for 24 h; 0.5% DMSO treatment was used as control. Briefly, cells were fixed with 3.7% formaldehyde (ThermoFisher Scientific, Waltham, MA, USA, cat. no. SF100-4) for 10 min and permeabilized with 0.2% Triton X-100 for 5 min. Cells were washed with phosphate buffered saline (PBS) and blocked with 3% bovine serum albumin for 1 h at room temperature and then incubated with rhodamine-phalloidin (1:500) (ThermoFisher Scientific, Waltham, MA, USA, cat. no. R37110) for 20 min. Cells were stained with MitoTracker Red CMXRos (1:1000) (Invitrogen, Waltham, MA, USA, cat. no. M7512) for 30 min. DAPI (1:1000) (ThermoFisher Scientific, Waltham, MA, USA, cat. no. D1306) was used to label the nuclei. Fluorescent images were captured with a Zeiss LSM 880 Confocal Microscope with ZEN 3.2 (blue edition) software (Carl Zeiss Microscopy, White Plains, NY, USA). The average relative fluorescence intensity was measured with the ImageJ (NIH). A one-way ANOVA test was performed to compare MitoTracker labeling in control cells versus treated cells.

Confocal microscopy was performed to evaluate peptide-RBCs binding in vitro. After 90 min of incubation with Rhd-labeled peptides, RBCs were washed, and nuclei were stained with Hoechst (1 μg/mL) (Sigma-Aldrich) and membrane was stained with CellMask Green (5 μg/mL) (Thermo Fischer Scientific). Images were taken with a Zeiss LSM900 with Airyscan 2 and analyzed with ZEN 3.2 (Blue edition) software (Zeiss, Oberkochen, Germany). Volume rendering was performed using IMARIS Software v9.3 (Bitplane, Zurich, Switzerland).