4 6 diamidino 2 phenylindole (dapi)

Cells were cultured in a 12-well plate, and FOXA2, RUNX2, COL1A1, t-ERK and p-ERK were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, permeabilized and blocked for 30 min in 0.05% Triton X-100 and 5% bovine serum albumin (BSA). Fixed cells were washed three times with PBS and incubated at 4 °C overnight with anti-FOXA2 (1:400; Cell Signalling Technology), RUNX2 (1:1600; Cell Signalling Technology), COL1A1 (1:500; Abcam, Shanghai, China), t-ERK (1:800; Cell Signalling Technology) or p-ERK (1:200; Cell Signalling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) at room temperature for 2 h and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. Samples were then observed and photographed under a fluorescence microscope (Leica).

Cells were cultured in induction medium in a 12-well plate and evaluated for RUNX2, SIRT7, and β-catenin using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany) as follows. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized, and blocked for 30 min in 0.05% Triton X-100 and 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 (1 : 1600; Cell Signaling Technology), SIRT7 (10 μg/ml; Abcam), or non-phosphorylated (active) β-catenin (1 : 1600; Cell Signaling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 120 min, and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 5 min. Samples were observed under a fluorescence microscope (Leica).

HK-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, United States). The ALR shRNA lentiviral and control shRNA lentiviral were purchased from Genechem Co. Ltd (Shanghai, China). The JC-1 fluorescence kit and Annexin V-FITC/PI apoptosis detection kit were obtained from Sigma-Aldrich (MO, United States). MitoSOX was obtained from Invitrogen (Thermo Fisher, MA, United States). 4,6-Diamidino-2-phenylindole was acquired from KeyGen Biotech. Co. Ltd (Nanjing, China). The ALR primer was designed by Sangon Biotech Co., Ltd (Shanghai, China). Total RNA extraction kit and its reverse transcription kit were obtained from Takara Biotechnology (Shiga, Japan). Anti-PINK1, anti-Parkin, anticytochrome c oxidase subunit IV (anti-COX IV), antitranslocase of outer mitochondrial membrane 20 (anti-TOMM20), antitranslocase of inner mitochondrial membrane 23 (anti-TIMM23), anti-P62, and anticleaved caspase-3 (CASP-3) were purchased from Abcam (Cambridge, United Kingdom). Anti-ALR and anti-LC3B were purchased from Thermo Scientific (Thermo Fisher) and Cell Signaling Technology (CST, United States), respectively. Antibeta actin (anti-β-actin) was from Tianjin Sungene Biotech Co., Ltd (Tianjin, China). Cell mitochondrial extraction kits and all secondary antibodies for fluorescent labeling or immunoblot analysis were from Beyotime Biotech Co., Ltd (Shanghai, China).

Tumor-bearing mice were sacrificed approximately 35 days after orthotopic xenotransplantation. After the intracranial xenograft tumors were harvested, pieces of the tumors were dissociated and subcultured in vitro, and other pieces were used for pathological examination. Frozen sections were stained with H&E, and 4',6-diamidino-2-phenylindole (KeyGen, Nanjing, China) was used for nuclear labeling. The sections were then observed under both a fluorescence microscope and a confocal laser scanning microscope (ZEISS, Germany).
For cell subculture, minced tumor tissue was washed with cold phosphate-buffered saline, digested with 0.02% trypsin and cultured in DMEM containing 10% fetal bovine serum (FBS, Gibco). After the cells had been serially subcultivated in vitro for approximately two weeks, fluorescence-activated cell sorting was used to sort suspensions of RFP⁺ tumor cells and EGFP⁺ stromal cells, and EGFP-expressing tumor stromal cells were collected. Then, the limiting dilution method and single-cell cloning techniques were used to re-purify EGFP-expressing cells with high proliferation abilities from the different animal models [23 (link), 38 (link)]. The EGFP-expressing tumor stromal cells from Models I, II and III were named EGFP⁺ tumor microenvironment cells 1 (^EGFP+TMEC1, ^EGFP+TMEC2 and ^EGFP+TMEC3, respectively).

TAECs cultured to 40–50% confluence on 1 cm culture plates were fixed and blocked, and they were then incubated with primary antibodies including anti‐VE‐cadherin (1:200; ab33168, Abcam, London, UK) and anti‐F‐actin (1:200; ab130935, Abcam, London, UK) overnight at 4 °C. Plates were washed and were then incubated with anti‐mouse or anti‐rabbit fluorescein isothiocyanate‐ and/or tetramethylrhodamine isothiocyanate‐conjugated secondary antibodies (Invitrogen, CA, USA). Cells were counterstained with 4′‐6‐diamidino‐2‐ phenylindole (KeyGen, Nanjing, China) to visualize nuclei and were observed using an inverted fluorescence microscope equipped with an Olympus Qcolor 3 digital camera.