Paraffin

During the 8th, 16th and 24th weeks of treatment, the mice were weighed and anesthetized with 3% pentobarbital sodium (30 mL/kg, Sigma-Aldrich Chemical Company, St. Louis MO, USA) dissolved in normal saline (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China) via an intraperitoneal injection. Next, venous blood was collected in heparinized tubes for biochemical assays. The bilateral kidneys of each mouse were removed, with one portion weighed and used for assay of inflammatory factors by reverse transcription quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The remaining part of the kidney was fixed by immersion with neutral formalin solution, dehydrated using gradient alcohol, cleared by xylene and embedded in paraffin (Thermo Fisher Scientific, San Jose, CA, USA). Next, 5-μm kidney tissue slices were prepared for histopathological observation.

Hepatic tissue and epididymal fat were preserved in a 10% formalin (Abcam, USA) solution overnight before being embedded in paraffin (Thermo Fisher Scientific, USA). 4 µm slices were prepared and subsequently deparaffinized with the xylene (Sigma, St Louis, USA) and concentration gradient of ethanol and rehydrated using deionized H₂O for two washes of 5 min. Furthermore, we stained with hematoxylin and eosin (H&E, Harris Hematoxylin and Eosin Y; Sigma, St Louis, USA) (21 (link)), the H&E-stained slides were inspected using a Leica Microsystems CMS GmbH (Wetzlar, Germany), and morphological pictures were analyzed using the SIS 3.2 software (Soft-Imaging System).

Specimens obtained via EBUS‐TBNA were fixed in 10% neutral buffered formalin solution for 20–28 h and embedded in paraffin (Thermo Fisher Scientific Inc.). Thin slides were prepared using formalin‐fixed paraffin‐embedded (FFPE) specimens cut to 3–4‐μm thickness with a microtome (Yamato Kohki Industrial Co., Ltd.), and histopathological examination was performed using the hematoxylin–eosin staining method. We collected data on histopathological analysis, including the presence of tissue cores, degree of blood contamination, and number of tumor cells on the slides. We defined a tissue core as a contiguous string of lung cancer tissues on a slide²⁵ (Figure 1A,B). Blood contamination was classified into three levels: low (no or few blood cells affecting diagnosis), moderate (blood cells obscure a part of the specimen, but pathological diagnosis is possible), and high (many blood cells make pathological diagnosis difficult)¹⁹, ²⁶ (Figure 1C–E). The tumor cell count was defined as the number of tumor cells on the slide.¹² Cells that were crushed and difficult to identify as tumor cells were not counted. Experienced pathologists at Yokohama City University Medical Center performed all pathological examinations. Pathologists were blinded to the EBUS‐TBNA method used to collect the specimens.

For histologic analysis, lungs from control or PRMT7 mutant mice were fixed in 4% (w/v) paraformaldehyde (PFA, Sigma-Aldrich, USA) overnight at 4 °C. After paraffin (Thermo, USA) embedding, tissues were sectioned at 5 µm. For immunohistochemical staining, sections were deparaffinized and rehydrated. Antigen retrieval was performed using the microwave method [17 (link)]. Sections were then pretreated with 0.1% Triton X-100 and blocked with 5% BSA for 2 h, followed by incubation with diluted primary antibodies in 3% BSA overnight at 4 °C, 1 h incubation with selected secondary antibodies, and counterstained with hematoxylin. Antibodies used for histologic analysis were listed in Table S3. H&E staining was performed as previously described [17 (link)]. Briefly, sections were deparaffinized and rehydrated, followed by staining in harris hematoxylin solution for 8 min. Then, differentiate in 1% acid alcohol for 30 s and counterstain in eosin-phloxine solution for 1 min. At last, dehydrate, clear, and mount.