Anti cd31

To isolate endothelial cells, lungs and brains were digested with collagenase A (1 mg/ml) and DNAse (100 μg/ml; Worthington Biochemicals) using gentleMACs C tubes and a gentle MACS Dissociator (Miltenyi Biotec). Homogenates were passed through a 40 μm cell strainer. Leukocytes were depleted using CD45 immunomagnetic beads (Miltenyi Biotec) followed by positive selection for endothelial cells using anti-CD31 or anti-CD146 MicroBeads (Miltenyi Biotec). Endothelial cells (>90% CD31⁺) were grown in medium containing Endothelial Growth Supplement (EGS; Sigma Chemical). Endothelial cells were challenged with T. gondii tachyzoites of the RH strain and the percentages of infection, the numbers of vacuoles and tachyzoites per 100 cells were determined using light microscopy by counting at least 200 cells per monolayer. The dendritic cell line DC2.4 (gift from Kenneth Rock, University of Massachusetts) was infected with PTG T. gondii. Extracellular tachyzoites were removed by extensive washing at 1 h and 18 h after challenge followed by i.v. administration into mice.

Conjugated antibodies against NANOG (phycoerythrin (PE) conjugated goat polyclonal (IC1997P), 1:200 dilution), OCT-3/4 (PE conjugated goat polyclonal (IC1759P), 1:200 dilution), and Nestin (PE conjugated mouse monoclonal (IC1259P), 1:200 dilution) were purchased from R&D Systems Europe Ltd (Abingdon, UK). Fluorescein isothiocyanate (FITC)-conjugated anti-CD90 (mouse monoclonal, 1:100 dilution) was purchased from Dianova GmbH (Hamburg, Germany).
All other antibodies used for cytofluorimetric analyses (FACS) were directly conjugated with FITC, PE, or APC: anti-CD31, antiCD-146/Muc-18, anti- CD29, anti-CD105, and anti-CD133 monoclonal antibodies (mAbs) (all from Miltenyi Biotec, Cologne, Germany); mAb anti-EpCAM (Biolegend, San Diego, CA). Isotype-matched FITC-, PE-, or APC- conjugated control mouse G (IgG) were from Miltenyi.

Lung tissue was cut into small pieces (1 mm³) and treated overnight at 4 °C with Trypsin/EDTA (0.25%; Gibco), penicillin/streptomycin (100 U/mL; 100 µg/mL), 2 mg/mL Collagenase A (Roche, Basel, Switzerland) and 0.04 mg/mL DNase (Sigma-Aldrich). Next, the suspension was diluted in DMEM containing 0.04 mg/mL DNase and penicillin/streptomycin, homogenized and filtered over two layers of gause filter (Sefar Nitex, Heiden, Switzerland). After centrifugation (500× g for 10 min) the pellet was resuspended in lysis buffer (15.5 mM NH₄CL, 1 mM KHCO₃, 0.01mM EDTA, 0.04 mg/mL DNase) for 10 min at 4 °C. The remaining pellet was resuspended in Small Airway Epithelial Cell Growth (SAGM) medium (PromoCell, Heidelberg, Germany) supplemented with penicillin/streptomycin, and kept on ice until use within 1–4 hrs. Cell suspensions were seeded into organoid cultures directly or EpCAM⁺ cells were isolated by anti-CD45 (#130-045-801, Miltenyi Biotec, Auburn, AL, USA) and anti-CD31 (#130-091-935, Miltenyi Biotec) coupled-microbeads and passed through LS columns (Miltenyi Biotec) according to the manufacturer’s guidelines. The flow-through was incubated with anti-EpCAM (CD326) microbeads (#130-061-101, Miltenyi Biotec) for positive selection with LS columns. Cells were resuspended in SAGM and kept on ice until use within 1–2 h.

Cultures of PF ADSCs and BC ADSCs at different passages (lower than passage 4) were phenotypically characterized following reference guidelines [32 (link), 33 (link)]. ADSCs obtained from PF- or BC-bearing patients were detached with 0.05% trypsin/EDTA (Thermo Fisher), washed with PBS, and 100000 cells were resuspended in 250 μL of PBS without Ca²⁺ and Mg⁺ (Euroclone, Pero, Italy) and incubated with antibodies directed against specific surface markers. Cells were incubated on ice for 30 minutes with antibodies anti-CD44 (BD Biosciences, San Jose, CA), anti-CD90 (Millipore, Massachusetts, USA), anti-CD34 (Miltenyi Biotec, Calderara di Reno, BO, Italy), anti-CD45 (BD Biosciences), anti-CD146 (Biocytex, USA), anti-CD31 (Miltenyi Biotec), anti-CD56 (Miltenyi Biotec), anti-CD105 (Serotec, Bio-Rad, Segrate, MI, Italy), anti-CD144 (R&D Systems, Minneapolis, MN, USA), anti-CD166 (BD Biosciences), anti-CD133/2 (Miltenyi Biotec), anti-CD73 (BD Biosciences), and anti-vascular endothelial growth factor 2 (VEGFR2; R&D Systems). Cells were pelleted, washed, and fixed in 4% paraformaldehyde (Sigma-Aldrich) for 20 minutes. Fluorescence-activated cell sorting (FACS) analysis was performed on a FACSVerse flow cytometer (BD Biosciences), equipped with the Cell Sweet software for data analysis.

The adrenal cortex was separated from the medulla under a dissecting microscope and was digested in 1.6 mg/ml collagenase I (Sigma-Aldrich) and 1.6 mg/ml BSA in PBS, for 25 min at 37°C while shaking at 900 rpm. The dissociated tissue was passed through a 22 G needle and 100 μm cell strainer and centrifuged at 300 × g for 5 min at 4°C. The cell suspension was washed in MACS buffer (0.5% BSA, 2 mM EDTA in PBS) and CD31⁺ and CD45⁺ cells were sequentially positively selected using anti-CD31 and anti-CD45 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer’s instructions. Briefly, pelleted cells resuspended in 190 μl MACS buffer were mixed with 10 μl anti-CD31 MicroBeads, incubated for 15 min at 4°C, washed with 2 ml MACS buffer, and centrifuged at 300 × g for 10 min at 4°C. Then, the cell pellet was resuspended in 500 μl MACS buffer, applied onto MS Columns placed on MACS Separator, and the flow-through (CD31^- cells) was collected. CD31⁺ cells were positively sorted from the MS Columns. The flow-through was centrifuged at 300 × g for 5 min at 4°C, and the pelleted cells were subjected to the same procedure using anti-CD45 MicroBeads, collecting the flow-through containing CD31^-CD45^- adrenocortical cells. CD45⁺ cells were positively sorted from the MS Columns.

ESCC cell lines-KYSE410 and KYSE510 were provided by Dr. Yutaka Shimada (Kyoto University). The primary CAFs, ESCC cells, TAMs, or ECs were isolated from fresh ESCC tissues (clinical stage: II) using magnetic-activated cell sorting (MASC) with anti-FSP (fibroblast specific protein, Miltenyi Biotec, Cat # 130-050-601), anti-CD326 (EpCAM, Miltenyi Biotec, Cat # 130-061-101), anti-CD14 (Miltenyi Biotec, Cat # 130-050-201), or anti-CD31 (Miltenyi Biotec, Cat # 130-091-935) microbeads according to manufacturer’s instructions. All cells were cultured in RPMI-1640 medium contained with 10% heat-inactivated FBS (Gibco) and 1% penicillin/streptomycin in a 37 °C humidified incubator under 5% CO₂.
The siRNA-based approach was applied to generate targeted genes-knockdown cells. Indicated siRNAs were transfected into primary CAFs using Lipofectamine 2000 reagent. For plasmid stable transfection, pcDNA 3.1-Flag plasmid contained AKT2 S128A or CCTα S315/319/323A mutant was transfected into CAFs. Subsequently, positive clones were selected for further experiments. Tansfection efficacy was evaluated using immunoblotting. Sequences of siRNAs were listed in Supplementary Table 3.

Dulbecco’s modified Eagle’s medium (DMEM), MesenPRO RS medium, DMEM-Ham’s F12 Nutrient mixture (DMEM-F12), fetal bovine serum (FBS), bovine calf serum (BCS), and l-glutamin (GlutaMax) were purchased from Gibco (Grand Island, NY, USA). SFEN, SFN, and IBR were purchased from LKT Laboratories (St. Paul, MN, USA). ERC, BITC, PEITC, AITC, indomethacin, methylisobutylxanthine (IBMX), dexamethasone, insulin, Oil Red O powder, MG132, and the antibody against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against PPARγ and n-acetyl-leu-leu-norleucinal (ALLN) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against C/EBPα and C/EBPβ were purchased from Cell Signaling Biotechnology (Beverly, MA, USA). Isopropyl alcohol was obtained from Amresco LLC (Solon, OH, USA). The MACS, anti-CD31, CD45 microbeads and MACS separation buffer were purchased from Miltenyl Biotec (Bergisch Galdbach, Germany).