Fig 2: Immune cell infiltration in the glioma microenvironment: MCs as potential modulators of SRGN expression in GBM(A) Immunohistochemical staining for CD163 in human low- and high-grade glioma TMAs. Representative TMA cores of low-grade glioma (upper left panel) and high-grade glioma (upper right panel) for CD163 expression. Selected areas from upper panels are magnified in the middle panel. TMAs included tumor tissue from high-grade gliomas (anaplastic gliomas and glioblastomas, n = 101) and gliomas WHO grade II (astrocytomas, oligoastrocytomas and oligodendrogliomas grade II, n = 87). Quantification of the CD163 expression levels (lower panel) shows significantly higher CD163 expression in the high-grade glioma TMA compared to the low-grade glioma. ***p < 0.001 (B) Immunofluorescence staining for CD3 and CD8 in human glioma TMAs. Representative staining's for CD3 (upper left panel), CD8 (upper middle panel) and CD3CD8 with DAPI staining (upper right panel). TMAs included tumor tissue from high-grade gliomas (anaplastic gliomas and glioblastomas, n = 101) and gliomas WHO grade II (astrocytomas, oligoastrocytomas and oligodendrogliomas grade II, n = 87). Quantification of the CD3 and CD8 expression levels (lower panel) shows no significant difference in the CD3+CD8+ cells between the low- and high-grade glioma TMA. Data is expressed as mean values + SEM and significance differences are indicated in the figure. ns = not significant. Scale bar = 200 µm (C) Spearman's rank correlation analysis between the number of infiltrating MCs and serglycin expression level, (Spearman's rho (?) = 0.2). LGG = low-grade glioma, HGG = high-grade glioma.

Fig 3: Significance of ERK pathway in SRGN-induced cell invasion and c-Myc stabilization. (A) Effects of SRGN-knockdown on mRNA expression of SRGN, CD44, c-Myc, CCND1 in ESCC cells. (B) SRGN-overexpressing ESCC cells were treated with trametinib (at indicated concentrations) for 24 h, and the SRGN mRNA expression level evaluated by RT-PCR. Cells treated with DMSO served as control. (C) SRGN-overexpressing ESCC cells were treated with 100 nM trametinib for 48 h, and then evaluated by invasion assay (upper panel, scale bar, 100 µm). The quantification is shown in the lower panel. (D) SRGN-overexpressing cells were treated with increasing concentrations of trametinib as indicated for 72 h. The expressions of members of the MEK/ERK pathway cascade and c-Myc were evaluated by western blotting. Untreated vector controls were included for comparison. (E) KYSE150-SRGN cells with or without trametinib (100 nM) treatment were incubated in 100 µg/mL cycloheximide (CHX) for indicated duration before western blotting analysis (left panel). The numbers below the c-Myc blots are the band intensities of c-Myc that were normalized against GAPDH and then expressed relative to that at 0 h time point. The relative c-Myc degradation rate is presented in the graph (right panel). (F) KYSE150 cells with SRGN-knockdown were subjected to CHX chase assay and the band intensities of c-Myc were quantified. CHX chase assay results of KYSE410 cells are shown in Figure S6A-B.

Fig 4: MDK binds to glycosylated SRGN. (A) Typical images of ESCC cells immunostained for SRGN (red) and MDK (green), and counterstained with 4',6-diamidino-2-phenylindole (DAPI) are shown in the left panel (scale bar, 10 µm). Line-scan profiles of immunofluorescence signals along the white arrows showed co-localization of SRGN and MDK. (B) Proximity ligation assay (PLA) for SRGN and MDK was performed on KYSE150 and KYSE410 cells. After ligation and amplification, the nuclei were counterstained with DAPI. Red spots (indicated by white arrows) represent the binding between SRGN and MDK proteins. Negative control was conducted by replacing SRGN antibody with mouse IgG. Scale bar, 10 µm. (C) Whole cell lysates of MDK-SFB transfected ESCC cells were immunoprecipitated with anti-FLAG M2 beads before immunoblotting for cellular SRGN and CD44. Western blotting showed that FLAG-fusion MDK co-precipitated with SRGN at ~250 kDa (red frames), and with CD44 at over 130 kDa. (D) Cell lysates and CM of FLAG-fusion SRGN-overexpressing (F-SRGN) and FLAG-fusion ?GAG-expressing (F-?GAG) cells were incubated with anti-FLAG M2 beads. The core protein of SRGN was predominantly precipitated in cell lysate and glycosylated SRGN was predominantly precipitated in CM. Western blots showed that MDK and MMP2 were bound to glycosylated SRGN, but not to SRGN core protein. MMP9 and CD44 were bound to both glycosylated SRGN and core protein. (E) Proteoglycans isolated from CM of KYSE150-SRGN cells were digested with the indicated enzymes before detection of SRGN using western blotting. (F) Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis of disaccharide products in the CM of KYSE150 cells expressing CON, SRGN, ?GAG and mGAG, with or without predigestion with chondroitinase ABC. Lanes Std1, Std2, and Std3 contained the standard markers. ?di-0S, ?di-6S, ?di-4S and ?di-SE in the CM of SRGN-expressing cells are indicated by red arrows.

Fig 5: Increased SRGN expression is accompanied by increased expression of a distinct set of mediators in both glioma cells and MCs(A) qPCR analysis for the expression of CXCL10, CXCL12 and TNF-a in the glioma cells after co-culture with LAD2 cells for 48 hours show a significant increase in expression in the co-cultured glioma cells as compared to not co-culturing with LAD2 cells. (B) qPCR analysis for the expression of CXCL1 and IL-6 in the LAD2 cells after co-culture with the glioma cells for 48 hours show a significant increase in expression in the co-cultured LAD2 cells as compared to not co-culturing with glioma cells. All the results are represented as increase in fold expression compared to the expression levels without co-culture. The experiments were performed 3 times, with triplicates in each case. Data is expressed as mean values + SEM and significance differences are indicated in the figure. GC = glioma cells, MC = LAD2 cells, GCMC = glioma cells co-cultured with LAD2 cells, MCGC = LAD2 cells co-cultured with glioma cells. ns = not significant, *p < 0.05, **p < 0.01 versus cells grown without co-culture.