Dpbs 1x

mDCs and pDCs were detached from the plates with DPBS 1X (Thermo Fisher Scientific, Waltham, MA, USA) + 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA). Then, cells were washed with DPBS 1X + 0.5% BSA (Sigma-Aldrich, St. Louis, MO, USA) and labeled with CD8a FITC, Ly6C FITC, CD11c APC-Vio770, CD45R(B220) PE-Vio770, CD11c PE, MHCII APC, CD80 FITC, and 7-AAD Staining solution (Miltenyi Biotec, Bergisch Gladbach, Germany). Flow Cytometer acquisition was performed using NAVIOS (Beckman Coulter, Brea, CA, USA). Flow cytometer analysis was performed using Kaluza Software 1.5 (Beckman Coulter, Brea, CA, USA).

Swelling test was performed on Matrigel, Matrigel with 3 mg/mL collagen type I, Matrigel with 6 mg/mL collagen type I, 3 mg/mL collagen type I (COL3MG) and 6 mg/mL collagen type I (COL6MG). The samples were incubated for 50 min to achieve gelation. After measuring the initial weight, the samples were maintained in deionized water for 24 h and the swollen weight was measured to calculate the swelling ratio. Degradation of the hydrogels by collagenase was measured. Collagenase type I (Worthington Biochemical Corporation, Lakewood, NJ, USA), was added to the DPBS (1x) (Thermo Fisher Scientific, Waltham, MA, USA) to a concentration of 1 U/mL and added to hydrogels maintained in DPBS. The weight loss of the hydrogels was measured at 0, 2, 6 and 18 h.

Total protein extracts were prepared as follows: Cells were trypsined and washed with cold DPBS 1X (ThermoFisher) and then were centrifuged at 200 × g for 10 min at 4 °C. Cell pellets were suspended in 50 µL of ice-cold lysis buffer [100 mM Tris-HCl, pH 7.4, 1% Triton, 0.25% sodium deoxycholate, 0.1% SDS, 300 mM NaCl, 1 mM EDTA, 1X PhosSTOP (Sigma), 1X Protease Inhibitor Cocktail (Sigma)]. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (ThermoFisher). Migration of an equal mass of total protein and of 5 µL PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa (ThermoFisher) was carried out on an 8% acrylamide denaturing gel. Proteins were transferred onto a nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad, city, state abbreviation if USA or Canada, country). The primary antibodies used against NRF1, p-AMPK, p-ACC and tubulin were mouse ab55744 (Abcam, Cambridge, United Kingdom), rabbit #07-681 (Merck), rabbit #11818 (Cell Signaling, Danvers, MA, USA), and mouse T5168 (Sigma), respectively. Incubation of the membrane with the primary antibodies was performed according to the manufacturer’s protocol. The secondary antibodies used were goat anti-rabbit #65-6120 and goat anti-mouse #62-6520 (Invitrogen). Detection of proteins was performed by chemiluminescence using Clarity Western ECL Blotting Substrate (Bio-Rad).

WT and Winnie mice were sacrificed, and their MLNs and PP were detached, cleaned from fat, and put in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA). To obtain a single-cell suspension, MLNs were smashed with DPBS 1X (Thermo Fisher Scientific, Waltham, MA, USA) + 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and passed through 40 µm cell strainers (Miltenyi Biotec, Bergisch Gladbach, Germany). The single-cell suspension from PP was obtained after digestion with collagenase type IV and DNase I (Sigma Aldrich, St. Louis, MO, USA) for 30 min at 37 °C on a rocking platform. The resulting single-cell suspension was pelleted by centrifugation, washed with DPBS 1X + 0.5 mM EDTA, and passed through 100 μm, 70 μm, and 30 μm cell strainers (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, cell pellets were washed with DPBS 1X + 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and labeled for cytofluorimetric analysis, according to the manufacturer’s instructions.

Following the collection of conditioned OM, the 3-D organoid suspensions were collected and suspended in VitroGel^® Cell Recovery Solution (TheWell Bioscience MS03-100), washed with DMEM/F-12 and pelletized by centrifugation at 600 xg for 6 min. Organoid pellets were snap frozen in liquid nitrogen and stored at -80 °C for downstream use. The EV-containing conditioned OM were then centrifuged in sequential order at 500 xg for 10 min, 3,000 xg for 10 min and 17,200 xg for 30 min and filtered through a 0.22 µm sterile filter (Millex^® SLGPR33RB) to remove residual cellular contaminants, large particles and protein aggregates. All centrifugation steps were performed at 4 °C. Bovine oviductal organoid-derived EVs were isolated from ~ 10 mL of conditioned OM (8 replicates/treatment group) using ultracentrifugation followed by size exclusion chromatography (SEC). Briefly, OM were subjected to two consecutive rounds of ultracentrifugation at 120,000 xg for 70 min in the Optima XE-90 Ultracentrifuge using the SW 55Ti rotor (Beckman Coulter A94471). Isolated EVs were further standardized by elution in 180 µL of sterile DPBS (1X) (ThermoFisher Scientific 14,190–144) using the Exo-spin™ mini columns (CELL guidance systems EX03-50) according to the manufacturer’s protocol. Isolated EVs were then stored at -80 °C for further characterization and molecular analysis.

TU-tagging experiments were performed on the following 1-week-old adult flies: Hand>LacZ;HA-UPRT, Hand>MblRNAi;HA-UPRT and Hand>Bru3;HA-UPRT. Briefly, flies were starved for 6 hr at 25°C and transferred to fresh food media containing 4TU (Sigma) at 1 mM. After 12 hr incubation at 29°C, about 30–40 flies of the described genotypes were dissected (removing head, wings, legs, ovaries and gut) in DPBS 1X (Gibco, Life Technologies), immediately transferred to Eppendorf tubes, and snap-frozen in liquid nitrogen. Total RNA isolation was performed as described above in TRIzol following the manufacturer’s instructions (ThermoFischer Scientific).