Pdgfr β

Animals were euthanized and perfused with saline solution 4 h (for inflammation and DNA DSB staining) or 3 days (for cell proliferation and vasculature staining) after ²¹²Pb-αVCAM-1 treatment. Inflammation, DNA DSBs, tumor cell proliferation, pericytes, microglia, and astrocytes were evaluated using primary antibodies against VCAM-1 (5 µg/mL, SoutherBiotech), γH2AX (2 µg/mL, Abcam), Ki67 (0.35 µg/mL, Dako), CD31 (5 µg/mL, PB Bioscience), platelet derived growth factor receptor beta (PDGFRβ) (2 µg/mL, Santa-Cruz), CD68 (1 µg/mL, Merck Millipore), and glial fibrillary acidic protein (GFAP) (3 µg/mL, Dako), respectively. Immunostaining protocols were performed as previously described.^{13 (link)} Tissue sections were examined at 20x magnification for VCAM-1 and at 40x for Ki67, γH2AX, CD31, PDGFRβ, CD68, and GFAP using a Leica DMi8 microscope. BM were identified by Hoechst 33342 counterstaining and through green fluorescent protein expression of MDA-231-Br cells. Whole brain images were obtained using Metavue software. For quantification of VCAM-1, CD31, γH2AX, and Ki67, three slices per animal were used and vessels, foci, and nuclei were automatically counted (ImageJ). Quantitative results of γH2AX and Ki67 staining are expressed as the percentage area of biomarker expression relative to the total tumor area. Vessel diameter was quantified as previously described.^{16 (link)}

Canagliflozin was provided by Mitsubishi Tanabe Pharma Corporation (Osaka, Japan). Streptozotocin and AICAR were purchased from Wako (Osaka, japan). Nicotinamide was purchased from SIGMA (St. Lewis, MO, USA). 2-Deoxyglucose (2-DG) was purchased from Tokyo Chemical Industries (Tokyo, Japan). Compound C was purchased from Calbiochem (CAS 866405-64-3). The cell culture reagents RPMI and DMEM were purchased from Nissui (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Biosera (Kansas City, MO, USA).
Antibodies were obtained from Cell Signaling Technology (pACC-11818S, ACC-3662S, pAMPK-4188S, AMPK-5832S, Cyclin D1-2922S, p-p70-9234S, p-70-2708S), R&D (PDGFRβ) and Santa Cruz (actin sc-47778 F1417, Pin-1sc46660 B0917).

Immunofluorescence staining was performed according to an established protocol^{9 (link),24 (link),25 (link)}. Kidney sections were fixed and stained with rabbit anti-collagen I antibody (Rockland Immunochemicals, Limerick, PA), rabbit anti-fibronectin antibody (Sigma-Aldrich, St. Louis, MO), or rabbit anti-α-SMA antibody (Abcam, Cambridge, MA) followed by Alexa-488 conjugated donkey anti-rabbit antibody (Invitrogen, Carlsbad, CA). For double immunofluorescence staining, kidney sections were fixed and stained with rat anti-CD45 (BD Biosciences, San Jose, CA) and platelet-derived growth factor receptor (PDGFR)-β (Santa Cruz Biotechnology, Dallas, TX) followed by appropriate secondary antibodies. Slides were mounted with medium containing DAPI. Fluorescence intensity was captured using a fluorescence microscope (Nikon Instruments Inc., Melville, NY). Quantitative evaluation was performed in a blinded manner by one observer using a NIS-Elements software. The fluorescence-positive area was reported as a percentage of the total high-power field (HPF) area^{4 (link),6 (link),38 (link)}.

EVs secreted by SD-MSCs from two different human donors (days 36-40) were lysed in RIPA buffer (Santa Cruz, Dallas, TX) and protein contents estimated using microBCA assay (Pierce, Rockford, IL) and western blot assays were performed us 10 μg of proteins. Antibodies: PDGFR-β (Santa Cruz, cat# sc-432, 1:200), LAMP2 (Thermo Scientific, cat# MA1-20798, 1:200), TIMP-1 (Chemicon, cat#AB800, 1:1000), CD90 (BD Pharmingen, cat#555596, 1:200), TIMP-2 (Chemicon, cat#AB801, 1:1000), CD9 (AbCam cat#ab2215) and CD81 (AbCam cat#ab79559).

Cells were washed two times with ice-cold PBS after treatment and lysed with ice-cold lysis buffer (0.3% CHAPS, 2 mmol·L⁻¹ EDTA, 10 mmol·L⁻¹ pyrophosphate, 10 mmol·L⁻¹ glycerophosphate, 40 mmol·L⁻¹ HEPES [pH 7.4], one tablet of EDTA-free protease inhibitors (Roche, USA) per 25 mL for 15 min. Then, we acquired the supernatant by centrifugation at 12 000 × g for 10 min. The supernatant was then incubated with primary antibody overnight at 4 °C. After that, a 50% slurry of Protein A + G Sepharose was added and incubated at 4 °C for another 2 h. The immunoprecipitates were washed 3 times with ice-cold PBS. Then, 50 µL of 1x SDS sample loading buffer was added, boiled for 10 min, and analyzed by immunoblotting. The following antibodies were used: PDGFR-β (Santa Cruz, 1:50, sc-374573), TLN1 (Abcam, 1:1 000, ab108480), FAK (CST, 1:1 000, 3285), goat anti-mouse IgG HRP (Invitrogen, 1:10 000, 31430), and goat anti-rabbit IgG HRP (Invitrogen, 1:10 000, 31460).