Dapi solution

FISH analysis was performed using RNAscope-Probe-HS-LINC00958 (Cat No. 478601) by Advanced Cell Diagnostics (CA, USA). Briefly, the TMA was dewaxed, dehydrated, pretreated with hydrogen peroxide for 10 min, and digested with protease. Next, the TMA was hybridized with the probe for 2 h at 40°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Cell Signaling Technology, MA, USA). The LINC00958 signal was analyzed using the TissueFAXS Spectra panoramic tissue multispectral imaging quantitative analysis system (TissueGnostics, Vienna, Austria). The tissues were classified into the following two groups based on the percentage of cells with the LINC00958 signal (LINC00958 signal-positive cells/total cells): low-expression group, < 5% of cells with LINC00958 signal; high-expression group, > 5% of cells with the LINC00958 signal. Next, the localization of LINC00958 in LUAD cells was analyzed using FISH, following the manufacturer’s instructions. The TMA was denatured at 80°C for 10 min and hybridized with the hybridization solution containing the probe at 37°C for approximately 10 h. Next, the samples were counterstained with DAPI solution (Cell Signaling Technology) and sealed with FixoGum Rubber Cement (EMS, Beijing, China) in the dark. The fluorescence images were captured under a fluorescence microscope (Zeiss Germany, Oberkochen, Germany).

DAPI staining and residual DNA quantification were used to evaluate the residual DNA which was an important index for dECM. For DAPI staining, the samples were fixed with 4% formaldehyde for 15 min and rinsed with PBS. DAPI solution (#4083, Cell Signaling Technology) at the concentration of 1 μg/mL was added, and the samples were incubated for 5 min at room temperature avoiding light. After PBS washing, the samples were coated with anti‐fade mounting solution and imaged under a fluorescence microscope (Eclipse Ti2, Nikon).
The content of residual DNA was quantified by a cell proliferation assay kit (C7026, Invitrogen) according to the manual. Native cells were washed with PBS, frozen, and stored at −80°C. The frozen cells and dECMs were thawed at room temperature and incubated in CyQUANT® GR dye/cell‐lysis buffer for 2–5 min, protected from light. The samples were measured using a fluorescence microplate reader with filters for 480 nm excitation and 520 nm emission maxima. The standard curve was prepared with λDNA (provided by the kit). The DNA content of dECM divided by the DNA content of native cells was defined as relative DNA percentage.

Frozen sections (4–8 μm) were stained with H&E. Sections for immunofluorescence staining were fixed with pre-cooled acetone (4 °C) for 10 minutes. After this, the slides were rinsed with PBS and blocked with 5% goat serum for 30 min. A 1:300 dilution of primary antibodies or fluorescently-conjugated antibodies was applied to the sections and incubated in a dark humidified chamber overnight at 4 °C, subsequently followed by incubation with Alexa Fluor secondary antibody at room temperature for 1 h for not fluorescently-conjugated antibodies. The nuclei were stained with DAPI solution (0.1 µg/ml; Cell Signaling Technology, Danvers, MA, USA). Immunofluorescence images were captured using Keyence all-in-one fluorescence-microscope BZ-X800 (magnification, ×200; ×400; ×1000; KEYENCE CORPORATION, Tokyo, Japan). BZ-X800 Analyzer software was adapted for the extraction and quantification of the imaging data.