Cell lifter

To determine the CFU of fresh stool and for inoculating low-density growth plates, we performed serial dilution, targeting 10⁻⁶–10⁻⁸ dilutions. This process was performed twice to create two serial dilution replicate sets. The 10⁻⁶–10⁻⁸ dilutions were inoculated (100μL) onto PRAS BRU solid medium plates and PRAS YCFAC-B solid medium plates in triplicate. The plates were incubated in the anaerobic chamber at 37°C for 120 hours. Photographs were taken of each plate, and colonies were counted for the plates in the countable range of 30–300 colonies (see S1 Fig for examples of CFU and high-density culture plates). CFUs from BIOME-Preserve tubes were assessed following the same protocol, except 10⁻⁴–10⁻⁶ dilutions were plated.
Bacterial colonies were carefully removed from plates with approximately 2,000 isolated colonies using the beveled edge of a cell lifter (08-100-240, Fisher Scientific, Hampton, NH) and transferred to a 2mL cryovial. Harvested cells from the three replicate plates from a single dilution replicate were combined into a single cryovial. 2mL of dilution blank were added to the cryovials, the cellular material was mixed via mechanical mixing, pipet mixing, and repeated inversion of the tube, and 1mL of material was transferred to a new 2mL cryovial. All cryovials were frozen immediately at -80°C; one of each duplicate tube was sent for WGS sequencing and analysis.

Bulk RNA-sequencing was applied to profile transcriptomic changes under 17β-estradiol treatment. BMEC-like cells were purified onto 6-well plates, and 24 h after subculture, 10 µM of 17β-estradiol or DMSO was prepared in EC medium lacking bFGF and RA and added to the cells. After 24 h, BMEC-like cells were washed once with DPBS, lifted using a cell lifter (Fisher Scientific #08–100-240), and collected via centrifugation. Resultant cell pellets were lysed in TRIzol reagent (Thermo Fisher Scientific #15,596,026) at room temperature for 10 min and stored at − 80 °C. Three biological replicates were collected per condition. Following the manufacturer’s guideline, RNA from each sample was purified using a Direct-zol RNA Miniprep kit (Zymo Research #R2051) simultaneously. Purified RNA samples were submitted to the Vanderbilt Technologies for Advanced Genomics core and sequenced on an Illumina NovaSeq6000. Raw sequencing reads were trimmed and aligned to human genome (GRCh38) with HISAT2 2.2.0 [42 (link)], and genomic features were obtained using the featureCounts function [43 (link)]. Differential gene expression and meta read counts were analyzed with edgeR [44 (link)]. Gene ontology enrichments analysis was performed on genes with p < 0.05 and fold change greater than 1.5 using WEB-based Gene SeT AnaLysis Toolkit (WebGestalt) [45 (link)].

Cells were grown to 50% confluency on 10 cm plates and treated with drugs or control for 24 h at 37 °C. Media was collected, cells washed in PBS and the supernatant was spun 500 g. Iced RIPA buffer (supplemented with a protease inhibitor cocktail (Sigma-Aldrich), 2 mM sodium orthovanadate and 50 mM NaF) was added to solubilize the cells (15 min, room temperature) at which point cells were collected using a cell lifter (Fisher Scientific). Supernatant cells were added to the RIPA buffer and combined with adherent cell fraction. Lysates were spun at 10,000g for 10 min at 4 °C, and supernatant was saved and quantified by bicinchoninic acid (BCA) assay (Pierce #23235). A measure of 30 μg of protein was loaded per well of a 15% SDS–polyacrylamide gel electrophoresis gel and transferred onto polyvinylidene difluoride membrane. The membrane was blocked in 5% dry milk (Genesee Scientific, #20-241) or 0.1% casein (Sigma C5890-500G). Primary antibodies were used at 1:1,000 dilution, and secondary horseradish peroxidase antibodies were used at 1:5,000 dilution or secondary fluorescent antibodies were used at 1:15,000. Fluorescent secondary antibodies were visualized using a LI-COR Odyssey scanner. Quantification of band intensity was performed in ImageJ and all normalizations were to the shown loading control. For uncropped western blots, refer to Supplementary Fig. 17.

Cells were grown to 80% confluency on 10cm plates at 37°C. Media was aspirated, cells washed in PBS, and the cellular monolayer was immersed in iced RIPA buffer (supplemented with a protease inhibitor cocktail (Sigma-Aldrich), 2 mM sodium orthovanadate, and 50 mM NaF). Following 15 minutes at room temperature (to better extract LC3 [9 (link)]), lysates were collected using a cell lifter (Fisher Scientific). Lysates were spun at 10,000g for 10 minutes at 4°C and supernatant saved and quantified by BCA assay (Pierce #23235). 10–30μg protein was loaded per well of a 15% SDS-PAGE gel and transferred onto PVDF membrane. The membrane was blocked in 5% dry milk (Genesee Scientific, #20–241). Primary antibodies were used at 1:1000 dilution, and secondary HRP antibodies were used at 1:5,000 dilution or secondary fluorescent antibodies were used at 1:15,000. Fluorescent secondary antibodies were visualized using a LI-COR Odyssey scanner. Quantitation of band intensity was performed in ImageJ and all normalizations were to the loading control displayed in the corresponding figure.

DCs were generated using bone marrow cells from C57BL/6NTac mice as previously described (20 (link)). In brief, cells were isolated from cleansed tibia and femur by flushing with cold PBS. The isolated cells were seeded at 3·10⁵ cells/ml in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% (v/v) FCS, 4mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 U/ml), and 50 μM β-mercaptoethanol (all products from Thermo Fisher Scientific). Complete medium was supplemented with 15 ng/ml granulocyte-macrophage colony stimulating factors (GM-CSF) for dendritic cell differentiation. For macrophage differentiation, isolated cells were seeded at 4·10⁵ cells/ml in complete medium supplemented with 30 ng/ml macrophage (M)-CSF. On day three and six new medium with GM-CSF or M-CSF was added, while on day eight the DCs were harvested by collecting all non-bound cells and for macrophages, cells were harvested by collecting all surface-bound cells using a cell lifter (Thermo Fisher Scientific).

Cells were seeded into the wells of 6-well plates (2 × 10⁶ cells/well) and allowed to adhere for ~16 h at 37 °C before the addition of drug(s). Media were replaced with fresh media containing 1 μM IAPa and cells were incubated at 37 °C for various amounts of time. At selected time points, media were aspirated and cells were washed two times with PBS and lysed with 150 μl RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma), 2 mM sodium orthovanadate and 50 mM NaF. Cells were collected using a cell lifter (Thermo Fisher, Waltham, MA, USA), transferred to an Eppendorf tube, passed through a pipet tip five times and incubated on ice for 10 min. The lysate was centrifuged at 14 000 × g for 5 min at 4 °C, and the supernatant (lysate) was collected and stored at −20 °C until western blot analysis.

Six-well tissue culture plates were coated overnight at 4 °C with the 5-clone monoclonal antibody cocktail containing IgG2a and IgG2b isotype monoclonal antibodies (100 μg/ml, 2 ml/well). Wells were washed twice with PBS prior to the addition of cells. BMDMs from WT and V-KO mice were cultured for 6 days on nontreated tissue culture plates and then harvested by incubating for 10 minutes at 4 °C and scraping with a Cell Lifter (Thermo-Fisher Scientific). Cells were washed, counted, and then added to either coated or uncoated wells at 3 × 10⁵ cells/well. Following an additional 24 hours of culture, RNA was isolated using the RNeasy Minikit (Qiagen, Germantown, MD, USA) for analysis by NanoString assay.