Extended Data Fig. 7: CXXC5 is the primary target gene of BMP signaling in DIPG subtype cells.
(a) qPCR analysis of CXXC5 mRNA expression in control or 2 hr-BMP4 (50 ng/mL) -treated indicated DIPG cells with or without pretreatment with 10 µg/mL cycloheximide (CHX) for 1 hr. n = 3 independent experiments. (b) FPKM values of CXXC5 in RNA-seq data of TT150630 DIPG cells treated with BMP4 at indicated timepoints (n = 2 independent experiments). (c) Immunoblotting analysis of CXXC5 and TUBULIN in indicated DIPG cell lines treated with BMP4 (50 ng/mL) at the indicated timepoints. The relative intensity of the CXXC5 protein level compared to TUBULIN is indicated. (d, e) qPCR analysis of CXXC5 relative expression in control and SMAD4 KD (d) or control, SMAD1 KD, and SMAD1/5 KD (e) DIPG cells treated with BMP4 (50 ng/mL) at indicated timepoints. n = 3 independent experiments. (f) Immunoblotting analysis for phosphorylated SMAD1/5, total SMAD1, CXXC5 and GAPDH proteins in the indicated cell lines. (g) qPCR analysis of ID1, SMAD7 and CXXC5 expression in SU-DIPG4 cells treated with BMP4 (50 ng/mL) at the indicated timepoints. n = 3 independent experiments. (h) Immunoblotting analysis for phosphorylated SMAD1/5, total SMAD1, CXXC5 and GAPDH proteins in SU-DIPG4 cell lines treated with BMP4 (50 ng/mL) at the indicated timepoints. (i) Immunoblotting analysis of CXXC5 and TUBULIN in indicated DIPG cell lines and PPC. SU-DIPG17 (1), TT150714 (2), TT150630 (3), TT160728 (4), TT150603 (5), TT160310 (6), TT151201 (7) and TT160518 (8). The relative intensity of the CXXC5 protein level is indicated. The experiments in c, f, h and i have been repeated three times with similar results. For a, d, e and g, data represents the mean ± S.D., statistical significance was calculated by two-tailed unpaired Student’s t-test.
