Phosphate buffered saline (pbs)

A pool of four shRNA targeting PIEZO1 cloned into a lentiviral vector (PLKO3.1‐CMV‐tGFP, MISSION tool, Sigma Aldrich) were used as previously described.
¹⁶ (link) In experiments performed with separated shRNA#1 and #2, clone used was described in Table S1. Lentiviral particle production was performed in HEK293T cells as previously described.
¹⁶ (link) Lentiviral supernatant was ultracentrifugated for 1.5 h at 100,000 g at 4°C; THP1 cells were transduced using a multiplicity of infection (MOI) of 10 in the presence of 8 μg/mL polybrene (Sigma Aldrich). Cells were washed twice in 1X PBS (Eurobio scientific) 24 h after transduction. At D4 post‐infection, GFP cells were sorted on a FACS Aria II instrument (Becton Dickinson) and cultured for 3 additional days. Cell counts, flow cytometry, gene and proteins analysis were performed at D4, D7, and D9.

T84 cells were seeded at a 250.10³ cells per well density in 24-well plates (Corning, Corning, USA) and incubated overnight with complete medium. Plates were set on ice 30 min before the beginning of the experiment. [⁶⁷Ga]Ga-AP747 was then added to the medium at a 0.1, 1, 10, 100, or 250 nmol/L concentration and cells were incubated for 2 h at 4 °C, in quadruplicate (n = 3). Incubation was stopped by removing the medium and washing cells twice with ice-cold PBS (Eurobio-scientific, Les Ulis, France). Finally, cells were treated with 1 mol/L NaOH, and the activity was measured using a gamma counter (Wizard 2480, Perkin-Elmer Waltham, USA). In order to assess non-specific affinity, an excess of non-radioactive apelin-F13A (final concentration 1 µmol/L) was added to selected wells.

Four-day-old siliques were collected on ice (extremities were removed, and the remaining part was cut into two pieces) and incubated for 1 h at 4 °C in the fixation buffer (1× PBS (Eurobio), 2% formaldehyde, and 0.1% triton X-100) after vacuum treatment (three times). Samples were then dehydrated using a series of increasing ethanol concentration in PBS (30%, 50%, 70%, 90%, 100%) at 4 °C (2 h each). Siliques were then stained using toluidine blue (0.01% in absolute ethanol), and transferred into a 2:1 followed by a 1:1 absolute ethanol–wax (wax: PEG400–1-hexadecanol, 9:1) solution for 2 h at 40 °C each, and finally transferred into a 1:2 solution (overnight at 40 °C). Samples were then incubated twice for 3 h in 100% wax solution at 40 °C before polymerization. Cross sections of 8 μm were finally cut using a Leica RM2165 microtome, and sample ribbons were placed on a drop of sterile water (Versol) on polyethylene slides, and left to dry overnight at 37 °C.
Immunolabelling using the JIM4 and JIM8 monoclonal antibodies (PlantProbes, Leeds, UK) was carried out as described in Macquet et al. (2007) (link). Samples were then observed using a confocal microscope (Leica TCS-SP2 AOBS, Leica Microsystems). Spectral bands from 498 to 567 nm were selected in order to specifically detect the Alexa Fluor 488 fluorescence.

Fecal microbiota was extracted by means of gradient purification under anaerobic condition (Freter chamber) as previously described³⁹ . Briefly, 2 g of thawed feces were diluted in 1X PBS (Eurobio), 0.03% wt/vol sodium deoxycholate, and 60% wt/vol Nycodenz (Sigma-Aldrich, St Louis, Mo) and loaded on a continuous Nycodenz based density gradient obtained by a freeze–thaw cycle. Fecal bacteria were obtained after ultracentrifugation (14,567 g for 45 min at + 4 °C; Beckman Coulter ultracentrifuge, swinging rotor SW28; Beckman Coulter, Fullerton, Calif) and washed 3 times in 1X PBS (Eurobio) and 0.03% wt/vol sodium deoxycholate. The final pellet was diluted in 8 mL 1X PBS–10% glycerol, immediately frozen in liquid nitrogen, and then stored at − 80 °C^{40 (link)}.