Fig 2: Cleavage of protein substrates by PSA.(A) Characterization of the proteolytic activity of PSA (0.2 µM) during 22 h incubation towards different protein substrates (1 µM each, except 0.5 µM semenogelin I) by SDS-PAGE and silver staining. Approximate molecular weights of the proteins are: PSA (28 kDa), semenogelin I (50 kDa), semenogelin II (63 kDa), fibronectin (220 kDa), galectin-3 (26 kDa), IGFBP-3 (30 kDa) and nidogen-1 (130 kDa). The lanes in which ~50% of the proteins were cleaved are bordered. (B) 1 µM MMP-3 (22 kDa), but not PSA, cleaved 1 µM plasminogen (88 kDa). Also 0.5 M fibronectin was incubated with 1 µM PSA as a control (SDS-PAGE with silver staining).

Fig 3: Degradation of nidogen-1 by PSA.PSA cleaved both mouse nidogen-1 in Matrigel and human recombinant nidogen-1. Mass spectrometry analysis identified nidogen-1 in the silver stained gel bands (arrows, left panel). Nidogen-1 bands of 140 kDa (A) and 110 kDa (B) disappeared and fragments of 90 kDa (C) and 55 kDa (D) appeared after 48 h incubation of diluted Matrigel with PSA at 37°C. Nidogen-1 cleavage by PSA in Matrigel was visualized by Western blotting with anti-nidogen-1 polyclonal antibody (middle panel). PSA (1 µM) cleaved recombinant human nidogen-1 (0.5 µM) into two fragments 85 kDa (arrow with *) and 55 kDa (arrow with **) during 20 h incubation at 37°C (right panel).

Fig 4: JQ1 drives chromatin accessibility changes in GC cells.a Overall chromatin accessibility changes of AGS cells following a 72 h treatment of JQ1 revealed by ATAC-seq analysis. b The location of the differentially accessible peaks were shown. c Motif-enrichment analysis of the differentially accessible sites. d The total number of differentially expressed genes of AGS cells after JQ1 treatment for 72 h revealed by RNA-seq analysis. e GO cluster analysis showed that the differentially expressed mRNAs were subjected to multiple GO terms. f The top 20 upregulated and top 20 downregulated genes after JQ1 treatment for 72 h demonstrated by RNA-seq analysis. g WB and qRT-PCR analysis of the mRNA and protein expression of NID1 in HGC27 and AGS cells treated with JQ1 (0, 200 nM, 500 nM, 1 µM, 2 µM, and 5 µM) for 72 h (mean ± SEM, ****p < 0.0001).

Fig 5: JQ1 effectively inhibits the proliferation of GC in vivo.a Twelve mice were divided into two groups and were treated with DMSO and JQ1, respectively. All mice and resected tumors were photographed after killing. b The weights and volumes of resected tumors from each mouse were recorded. c The body weights of each mouse were recorded. d The protein expression of NID1 of the resected tumors revealed by WB analysis. e The mRNA expression of NID1 of the resected tumors revealed by qRT-PCR analysis (mean ± SEM, **p < 0.01). f Schematic model for how JQ1 regulated the RUNX2/NID1 signaling via altering chromatin accessibility in GC cells.