Hpa029901

Paraffin-embedded synovial tissues of RA were sectioned, deparaffinized, and antigen-retrieved with 0.01 M citric acid of pH 6.0. The clinical background of the patients is shown in Supplementary Table 3. Immunohistochemistry was performed with Sox4 antibody (HPA029901, rabbit polyclonal, Sigma Aldrich, 1:100) and detected with EnVisionTM Detection System (DAKO). ELS formation (Score E) and the presence of Sox4-positive cells within the infiltrating cell population without ELSs (Score SN) or with ELSs (Score SE) were assessed by a semi-quantitative four-point scale (none, 0; mild, 1; moderate, 2; and severe, 3). The total Sox4 expression score corresponds with the sum of the SN and SE scores. Triple immunofluorescence staining was performed with CXCL13 antibody (BCA1, rabbit polyclonal, Thermo Fisher, 1:100), Sox4 (HPA029901, rabbit polyclonal, Sigma Aldrich, 1:30), CD3 (Clone PS1, mouse monoclonal, Leica Microsystems, 1:200), CD4 (Clone 1F6, mouse monoclonal, Leica Microsystems, 1:100), CXCR5 (Clone 51505, mouse monoclonal, R&D Systems, 1:1000), and PD-1 (Clone NAT105, mouse monoclonal, Abcam, 1:50) and detected with TSA Plus Fluorescence Kit (PerkinElmer, Inc.). Fluorescence imaging analysis was performed using the FSX100 Fluorescence Microscope (Olympus).

CUT&RUN coupled with high-throughput DNA sequencing was performed using antibodies targeting SOX4 (Sigma, Catalog: HPA029901, Lot: D116454), SOX11 (Sigma, Catalog: HPA000536, Lot: BG117774), and Cutana pA/G-MNase (Epicypher) according to the manufacturer’s protocol. Briefly, SMS-KCNR cells (5.0 × 105 per reaction) were washed and incubated with activated Concanavalin A beads for 10 min at room temperature. Cells were then resuspended in antibody buffer containing 0.01% digitonin, 1 mL of each antibody was added to individual cell aliquots, and tubes were rotated at 4 °C overnight. The following day, targeted chromatin digestion and release was performed with 2.5 mL Cutana pA/G-MNase and 100 mM CaCl₂. Retrieved genomic DNA was purified with the MinElute PCR purification kit and eluted in 10 mL of buffer EB. Sequencing libraries were prepared with the automated Swift 2S system, followed by 100bp-PE sequencing with Novaseq 6000.