Fig 2: Expression and localization of RUNX3 in tongue SCC. (A and B) Western blot analysis of the relative protein expression levels of RUNX3 in three tongue SCC tissues and paired non-cancerous tissues. **P<0.01. (C) Immunofluorescence of RUNX3 in tongue SCC cell lines SCC25 and Cal27; low expression was detected in the cytoplasm (magnification, ×200). Control groups were incubated with PBS instead of first antibody. (D) Immunochemistry of RUNX3 in tongue SCC cell lines SCC25 and Cal27 (left panel, magnification, ×100; right panel, magnification, ×200). The majority of staining was detected in the cytoplasm, but a number of individual cells presented with nuclear staining (black arrow). N, non-cancerous tissue; RUNX3, runt-related transcription factor 3; SCC, squamous cell carcinoma; T, tumor tissue.

Fig 3: Genome-wide analysis of Runx2 and Runx3 association profiles in chondrocytes.a Genome-wide distribution of Runx3-associated regions relative to transcriptional start sites (TSS) with Genomic Regions Enrichment of Annotations Tool (GREAT) gene ontology (GO) and MGI expression annotations of Runx3 peaks showing the top five enriched terms. b Enriched motifs determined by Runx3-FLAG ChIP-seq. c Schematic diagram to identify candidate genes by ChIP-seq and RNA-seq using primary SFZ cells obtained from Cntl and cKO mice. d Genome-wide distribution of Runx2-associated regions relative to TSSs, and top five enriched terms identified by GREAT GO of Runx2 peaks. Left upper panel, primary chondrocytes treated with vehicle; left lower panel, inflamed chondrocytes exposed to 1 ng per mL IL-1β; right panels, overlap of Runx2-FLAG ChIP-seq peaks between primary and inflamed chondrocytes. e Enriched motifs determined by Runx2-FLAG ChIP-seq. f Enrichment of TG(T/C)GGT and poly-A motifs recovered de novo from Runx2 and Sox9 peak regions. The upper panels show motif logos, displaying nucleotide frequencies (scaled relative to the information content) at each position. Lower panels show enrichment levels of TG(T/C)GGT and poly-A motifs mapped to Runx2 and Sox9 peaks, whereby x and y axes represent the distance from mapped motifs to the peak center and frequency of mapped motifs, respectively. g Signal intensity plots of ChIP-seq data for Sox9, H3K4me2, H3K27ac, RNA polymerase II (polII), and rabbit immunoglobulin G (IgG) controls in ±1 kb from Runx2-FLAG peaks. Normalized mean signal intensity for the following ChIP-seq reads are shown in the top panels. TES, transcriptional end site. h Schematic of identification of anti-apoptosis-associated candidates among downregulated genes by homozygous Runx2 knockout in primary chondrocytes. i Schematic of identification of candidate transcriptional target genes of Runx2 in inflamed chondrocytes.

Fig 4: Protein expression of GATA3, STAT6, T-bet and Runx3 in the lung tissues of mice. (A) Western blotting images and (B) semi-quantification of the protein expression of GATA3, STAT6, T-bet and Runx3. Data are expressed as the mean ± SD; n=6; *P<0.05 vs. Control; ∆P<0.05 vs. PM2.5; #P<0.05 vs. OVA. OVA, ovalbumin; PM2.5, 2.5 µm particulate matter; GATA3, trans-acting T cell-specific transcription factor GATA-3; T-bet, T-box transcription factor TBX21; Runx3, runt-related transcription factor 3.

Fig 5: RUNX3 accounts for down‐regulation of SIRT1 in H. pylori‐infected gastric cells. A, The scheme of the putative RUNX3‐binding site in the SIRT1 promoter region. B, ChIP assay of RUNX3 directly binding to the promoter of SIRT1 (n = 3). C–E, Luciferase activities of different SIRT1 promoter constructs in cells transfected with RUNX3‐expressing vector (C), siRNAs targeting RUNX3 (D) or Runt domain mutant RUNX3‐expressing vector (E). Data from 3 independent experiments are presented as mean ± SD. F and G, The qRT‐PCR analysis of RUNX3 (F) and SIRT1 (G) mRNA levels in gastric cells transfected with RUNX3‐expressing vector and infected with Hp26695 for 3 h. Data from 3 independent experiments are presented as mean ± SD. H, Western blot analysis of RUNX3 and SIRT1 protein levels in gastric cells transfected with RUNX3‐expressing vector and infected with Hp26695 for 3 h. The mean values are indicated (n = 3). * represents P < 0.05 and **** represents P < 0.0001