4 6 diamidino 2 phenylindole (dapi)

Human intestinal organoids were harvested using Cell Recovery Solution (Corning) from Matrigel. The organoids were treated with a blocking solution (3% bovine serum albumin solution in PBS). They were then fixed and permeabilized using Fixation/Permeabilization Kit (BD Biosciences), after which they were stained with BODIPY 493/503 (Thermo Fischer Scientific), Alexa Fluor 568 Phalloidin (Thermo Fischer Scientific), and DAPI (Dojindo) according to the manufacturers’ instructions. BODIPY, Phalloidin, and DAPI were used to counterstain neutral lipids, F-actin, and DNA, respectively.

Tumor tissues were collected from each mouse on Day 8 or 9 after treatment, dissociated into single cells by using a Tumor Dissociation Kit and a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). In cell mixtures, leukocytes positive for CD45 were isolated with mouse TIL (CD45) microbeads (Miltenyi Biotec) by using an OctoMACS Separator (Miltenyi Biotec). After washing and filtration, cells were blocked with Mouse BD Fc Block (BD Biosciences, San Jose, CA, USA), and stained with the antibodies listed in S1 Table and with DAPI (4′,6-diamidino-2-phenylindole, Dojindo, Kumamoto, Japan). The gating strategy is shown in S1 Fig. To detect IFN-γ-producing activated CD8⁺ T cells, we incubated CD45⁺ cells with RPMI1640 containing with 10% FBS, PMA/Ionomycin (Sigma, St. Louis, Missouri, USA), and BD GolgiPlug (BD Biosciences) for 4 h at 37 °C, and then stained them with antibodies against CD45, CD3 (Thermo Fisher Scientific, Waltham, MA, USA), CD4, and CD8 (BD Biosciences). The cells were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) and then stained with antibody against IFN-γ or GzmB (BD Biosciences). The cells were sorted with a BD FACS AriaII SORP or LSRFortessa X-20 (BD Biosciences) and the data were analyzed by using FlowJo v10 (BD Biosciences) or Cytobank (Cytobank, Inc., Santa Clara, CA, USA).

Cells were cultured in 96-well plates and treated with various concentrations of PDGF-BB, TGF-β, and/or TNF-α in combination with palbociclib and/or etanercept for 2 days. Cultured cells were fixed with 4% paraformaldehyde for 15 min at RT and then incubated in 2% BSA in PBS-T for 60 min at RT. After the cells were washed with PBS-T, they were incubated for 2 h at RT with a primary Alexa Fluor 488-conjugated anti-PDGFRA(Tyr849)/PDGFRB(Tyr857) antibody (1:20; Bioss, Boston, MA, USA) and a primary APC-conjugated anti-cadherin-11 antibody (1:80; BioLegend). Nuclei were stained with DAPI (1:1000; Dojindo). The fluorescence intensities of pPDGFRαβ and CDH11 staining were measured using a microplate reader (INFINITE M1000 PRO; Tecan Trading AG, Switzerland) and normalized based on the DAPI staining intensity.

Cultured endometrial epithelial cells were fixed in 4% paraformaldehyde for 5 min at room temperature. After permeabilization and blocking with 5% normal goat serum, the cells were treated simultaneously with two antibodies (anti-CK18 antibody,1:100; anti-vimentin antibody, 1:100) at 4 °C overnight. After rinse, the specimens were treated with secondary antibodies (Alexa Fluor 594 and Alexa Fluor 488; 1:1000, Molecular Probes, Invitrogen, Carlsbad, CA) and nuclei-stained by DAPI (1 µg/mL; Dojindo, Kumamoto, Japan). Stained specimens were observed using either a conventional fluorescence microscope (Olympus, Tokyo, Japan) or inverted confocal laser microscope (LSM510; Carl Zeiss, Oberkochen, Germany) with an appropriate set of excitation and emission filters. The samples in which the population ratio of CK18+ cells (the number of CK18+ cell/the number of total cells) was >80% were used for subsequent DNA methylation analysis (Supplementary Table S1).

Sections (4 μm) of the intestine from WT and Ager^−/− pups were stained with hematoxylin and eosin^{42 (link)}. The intestine was incubated with 4′,6-diamidino-2-phenylindole (DAPI, Dojindo; Kumamoto, Japan, 1:2,000) or polyclonal rabbit anti-RAGE antibody (1:1,000; AB5484, Millipore, Billerica, MA) and washed with 0.3% Triton X-100 in PBS. They were then incubated with Alexa Fluor 488- (1:200, Invitrogen Molecular Probes).
RAGE expression in the intestine of wild-type mouse embryos (18.5 dpc) (4 μm sections from GenoStaff Inc, Tokyo, Japan) were examined using the polyclonal rabbit anti-RAGE (1:1000), Alexa Fluor 488, and DAPI. Images were taken with the EVOS FL Cell Imaging System (ThermoFisher Scientific, Yokohama, Japan).

To visualize and compare the binding specificity of each selected aptamer, fluorescence images of various cell lines bound with the aptamer were observed by fluorescence microscopy (Eclipse Ti, Nikon) or confocal laser microscopy (FV3000 or FV10i-DOC, Olympus; A1R+ Confocal Microscope, Nikon). Each cell line was cultured on an 8-well chamber slide (Corning) and incubated with 250 nM Alexa 488-labeled aptamer, either on ice or at 37°C for 30 min. After the incubation, the supernatant was removed and the cells were washed with ice-cold washing buffer three times. The cells were fixed with 4% formaldehyde (Nacalai Tesque, Kyoto, Japan) for 10 min at room temperature, stained with DAPI (Dojindo Laboratories, Kumamoto, Japan), and mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA). In the confocal imaging, prior to the treatment with the aptamer, the cells were incubated at 37°C for 30 min in the presence of 75 nM LysoTracker Red DND-99 (Molecular Probes).