Dmi3000b microscope

All cell culture images were acquired on a Leica DMI3000B microscope (Leica, Wetzlar, Germany), using a 5x, 10x or 20x objective. pCAG-YFP was used in place of pCAG-GFP to induce dGBP1-TagBFP stability in order to avoid fluorescence bleedthrough from brightly fluorescent GFP signals into the TagBFP channel.

CCK-8 and plate colony formation assays were performed to evaluate the proliferative ability of the cells^{22 (link),24 (link)}. The cells were seeded in 24-well plates overnight and then transfected with miR-NC, miR-129-5p or miR-129-5p plus pcDNA3.1-Wnt5a. GBM stem cells or monolayer cells were trypsinized and seeded in 96-well plates at a confluence of 2000 cells per well per 100 μL of stem cells or 10% FBS supplemented DMEM. Absorption of the cells was measured at different indicated time points using the CCK8 kit (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s instructions. An EdU imaging kit (Life Technologies) was used to determine DNA synthesis of cells grown on coverslips in a 24-well dish after appropriate TMZ treatments. Immunostaining and EdU assay results were visualised using a Leica DMI3000B microscope. EdU-positive cells were manually counted.

Synovial tissues were fixed in 10% neutral formalin, embedded in paraffin, deparaffinized, hydrated with gradient alcohol, and then transversely sectioned for routine H&E staining. Krenn’s synovitis scoring system (grades 0–9) was used to quantify pathological changes in the synovium by observers who were blinded to the experimental groups (Krenn et al., 2006 (link)). The sum of the synovitis score was as follows: 0–1, no synovitis; two to four, low-grade synovitis; and five to nine, high-grade synovitis.
Sirius red and Masson staining were carried out according to the instructions of the kit (Solarbio, Beijing, China). Approximately 5 μm-thick synovial tissue sections were observed under a Leica DMI-3000B microscope (Leica, Germany). The degree of synovial fibrosis was evaluated by calculating the synovial fibrosis score and percentage of collagen I-positive areas with ImageJ (Version 1.74; National Institutes of Health, United States, available at http://rsbweb.nih.gov/ij/). The synovial fibrosis scoring system was as follows: absent, 0; mild, one; and diffuse, 2 (Joronen et al., 2004 (link); Liao et al., 2021 (link)).