Fig 2: CD109 Plays an Essential Role in Clonogenicity, Tumor Initiation, and Radioresistance in GBM(A) Western blotting of CD109 in GSC267 cells infected with shNT or with shCD109–1 and shCD109–2.(B) Neurosphere formation efficiency in GSC185A clones. ***p < 0.001. See also Figure S5A.(C) Representative H&E staining of xenograft tumor derived from GSC267 cells stably transduced with shNT or shCD109. Arrows (right) show the injection site. The inset image (left) shows the core tumor area.(D) Cell-cycle analysis of irradiated GSCs (6 Gy). The percentage of cells in the G2/M phase is indicated within each cell-cycle plot.(E) Immunofluorescence images of ?-H2AX foci in shNT and shCD109 cells (left). Bars indicate the number of foci 24 h after IR. Black bars represent shNT; gray bars represent shCD109. Error bars indicate SEMs. *p < 0.05 and **p < 0.005.(F) Annexin V and propidium iodide staining in GSC267 clones. The results are representative of 3 independent experiments.(G) Kaplan-Meier survival curves for mice implanted with GSC20 cells with or without fractioned intracranial radiation (2.5 Gy, Å ~4).

Fig 3: CD109/EGFR/STAT3 signalling may response for cell migration, proliferation and maintenance of CSC phenotype.Western blotting for EGFR expression and phosphorylation of STAT3 on a panel of human cervical cancer cell lines (a), sorted CD109(-) and CD109( + ) cells in C4-1 and CaSki lines (b), NTC siRNA or CD109 siRNA-knockdown cells in C4-1, CaSki and SiHa lines (c) and CaSki control or Q4-CD109-KO cells, SiHa control or L65-CD109-KO cells (d). Western blotting for phosphorylation of STAT3 on sorted CD109(-) and CD109( + ) cells with/without anti-EGFR treatment (e). Representative images of spheroids formed by sorted CD109( + ) CaSki cells treated w/o EGFR (magnification, ×4, ×10). The bar graph (below) represents the mean number of spheroids from each well under the microscope, and error bars represent SD, *P = 0.0021 (f). Representative images of spheroids formed by NTC siRNA or STAT siRNA-knockdown CaSki CD109( + ) cells (magnification, ×4, ×10). The bar graph represents *P = 0.0030 (g). Western blotting for stem cell markers on sorted CD109(-), NTC siRNA or STAT siRNA-knockdown CD109( + ) CaSki cells (h).

Fig 4: siRNA-mediated CD109 knockdown suppressed migration, cell proliferation and sphere-forming ability.a Western blotting for confirmation of CD109 siRNA knockdown in C4-1, CaSki and SiHa cells. b A representative image (left) and quantification bar graph (right) of migrated NTC siRNA or CD109 siRNA- knockdown cells (crystal violet). *PC4-1 < 0.0001, PCaSki < 0.0001, PSiHa < 0.0001, using the Student’s t test. c Proliferation analyses of NTC siRNA or CD109 siRNA-knockdown cells. *PC4-1 = 0.0274, PCaSki < 0.0001, PSiHa = 0.0006. d Representative images of spheroids formed by NTC siRNA or CD109 siRNA-knockdown cells (above, magnification, ×4, ×10). Bar graph (below) represents the mean number of spheroids from each well under the microscope, and error bars represent SD, *PC4-1 = 0.0096, PCaSki = 0.0020, PSiHa < 0.0001.

Fig 5: CD109-GP130 interaction promotes activation of the STAT3 pathway.(A) qRT-PCR analysis of IL6R and IL6ST mRNA levels in GSCs. Data are presented as mean ± SD. (B and C) Western blot analyses of p-STAT3 levels in CD109-silenced and nontargeted control GSCs after stimulation with IL-6 cytokine (50 ng/mL) for 15 and 30 minutes. (D) qRT-PCR analysis of IL6ST mRNA levels in CD109-silenced and nontargeted control GSCs. Data are presented as mean ± SEM. *P < 0.05; ****P < 0.0001, unpaired 2-tailed t test. (E) Western blot analysis of GP130 expression after CD109 silencing in GSCs of different glioblastoma subtypes. (F and G) Western blot analyses of CD109 expression and p-STAT3 levels in GP130-silenced and nontargeted control GSCs. (H) Co-IP analysis of CD109. GSC whole-cell extracts were subjected to immunoprecipitation with an anti-GP130 antibody or appropriate IgG control followed by Western blotting with an anti-CD109 antibody. Inputs are indicated. (I and J) Representative micrographs of PLA in GSCs using anti-CD109 and anti-GP130 antibodies. Appropriate IgG controls served as negative controls. Red indicates specific interaction signal. Nuclei were counterstained with DAPI (blue). Scale bar: 20 µm. (K and L) Quantification of PLA signal per cell (DAPI). Data are presented as mean ± SD. **P < 0.01; ****P < 0.0001, 1-way ANOVA with Tukey’s multiple comparisons test. Data are from n = 3 (A–G) and n = 2 (H–L) independent experiments. Representative Western blots are shown, where ß-tubulin served as a loading control.