Extended Data Fig. 2: High expression of CHRDL1 contributes to the low activity of BMP signaling and tumor progression in this DIPG subtype.
(a) Kaplan–Meier survival curves for DIPG patients in the UCSC xena patient cohort27, separated into CHRDL1 high(n = 8 patients) and CHRDL1 low(n = 9 patients) survival groups. Log-rank test was performed. (b) qPCR analysis of CHRDL1 expression in indicated Ctr or CHRDL1 KD cells. n = 3 independent experiments. (c) Immunoblotting analysis of p-SMAD1/5 and SMAD1 in TT150714 and SU-DIPG17 (Ctr or CHRDL1 KD DIPG cells) with or without BMP4 (25 ng/mL) for 2 hr. (d) qPCR analysis of BMP signaling response genes mRNA expression in Ctr and CHRDL1 KD TT150714 and SU-DIPG17 cells with or without BMP4 (25 ng/mL) treatment. Data represents the mean ± S.D., statistical significance was calculated by two-tailed unpaired Student’s t-test, n = 3 independent experiments. (e) Neural sphere formation of indicated cell lines (Ctr or CHRDL1 KD DIPG cells) for 10 days (n = 3 independent experiments). (f) Neural sphere counts from (e). (g) The normalized bioluminescence activity was plotted and the statistical difference between TT150630 Ctr and CHRDL1 KD groups was significant (n = 5 mice in Ctr and n = 5 mice in CHRDL1 KD). (h) Representative bioluminescence images from animals implanted with 5 ×105 Ctr (n = 5 mice) or CHRDL1 KD(n = 5 mice) of luciferase-GFP engineered-SU-DIPG17 cells in the pons at day 238. The heatmap superimposed over the mouse heads represents the degree of photon emission by DIPG cells expressing firefly luciferase. (i) The normalized bioluminescence activity was plotted and the statistical difference between SU-DIPG17 Ctr and CHRDL1 KD groups was significant (n = 5 mice in Ctr and n = 5 mice in CHRDL1 KD). (j) Kaplan–Meier analysis from animals implanted with SU-DIPG17 cells with (n = 5 mice) or without (n = 5 mice) CHRDL1 KD in the pons. Log-rank test was performed. (k) Immunofluorescence of pons section from animals implanted with TT150630 cells with or without CHRDL1 KD for anti-human nuclear antigen (HNA), OLIG2 and GFAP. Scale bars, 50 µm. This figure represents 9 independent tissues. (l) Quantification of OLIG2-positive cells in all tumor cells (HNA positive) from animals implanted with TT150630 cells with or without CHRDL1 KD. n = 9 independent tissue samples. For g and i, boxplots define the interquartile range (IQR) split by the median, with whiskers extending to the most extreme values within 1.5 × IQR beyond the box, statistical significance was calculated by two-tailed unpaired Student’s t-test. The experiments in c have been repeated three times with similar results. For b, f and i, data represents the mean ± S.D., statistical significance was calculated by two-tailed unpaired Student’s t-test.
