Fig 2: CHRDL1 is associated with worse survival in glioma and (A) An unfavourable prognosis in (B) urothelial and (C) renal cancer. According to data from the human protein atlas [37] (proteinatlas.org). * p < 0.05; *** p < 0.001; log-rank p-value for Kaplan–Meier plot. Image Credit: Human Protein Atlas.

Fig 3: Point-plots of Taqman-based gene expression of GS-5 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and measurement of (A) CHRDL1, (B) OLIG2, (C) SOX2, (D) SOX9, (E) ZEB1, (F) ZEB2, (G) CD44, (H) NES, (I) GFAP, (J) NEFL, (K) MAP2 and (L) RBFOX3 expression. The data are the summary of three experiments performed in triplicates. * p < 0.05; *** p < 0.001; **** p < 0.0001; ns: not significant; unpaired t test with Welch’s correction; white dot: shCtrl, red dot: shCHRDL1.

Fig 4: CHRDL1-depletion activates BMP4-signaling. (A,B) Western Blot of (A) NCH644 treated with 20 ng/mL recombinant human BMP4 for 24 or 48 h or (B) NCH644 shCtrl and shCHRDL1. (C,D) Sphere formation assay of (C) NCH644 and (D) GS-5 shCtrl or shCHRDL1 GSCs and treatment with 25 ng/mL recombinant BMP4 or solvent for 7 days after seeding of 500 and 1000 cells per well. * p < 0.05; **** p < 0.0001; two-way-ANOVA with Sidak’s multiple comparison tests; white dot: shCtrl, red dot: shCHRDL1.

Fig 5: CHRDL1 depletion blocks stemness in GSCs. (A) Western Blot of NCH644 and GS-5 GSCs after stable transduction with a non-targeting shRNA (shCtrl) or specific shRNA targeting CHRDL1 (shCHRDL1) and (B) Quantification of several independent experiments. (C,E,F) Log-fraction plots of the limited dilution model of data from (C) NCH644 shCtrl (black) and NCH644 shCHRDL1 (red) GSCs 7 days after seeding the cell in a dilution series from 1024 to 8 cells and analyzing the data using ELDA software [33]. (D) Representative microphotographs of NCH644 and GS-5 with and without CRHDL1-depletion. Scale bar: 200 µm. (E,F) Data from GS-5 GSCs (E) 7 days and (F) 14 days after seeding of 2048 to 16 cells. **: p < 0.01, ***: p < 0.001 ****: p < 0.0001.