Skip to main content
. 2022 Oct 11;82(24):4586–4603. doi: 10.1158/0008-5472.CAN-22-2108

Figure 2.

Figure 2. SOX9 anticorrelates with MYC and marks quiescent cells in Group 3 patients with MB. A, Genes with higher ATAC-seq peaks in GFP-positive GTML-pSOX9-GFP cells significantly overlap with a quiescent signature (Fishers exact test; P < 0.05). B, Boxplot comparing the ssGSEA scores for a quiescent NSC signature between the groups from C, indicating that Sox9 high GTML cells appear significantly more quiescent than MYCN-high and intermediate cells (Welch t test; P < 0.05). C, Single-cell sequencing of GTML cells indicates that there exist three populations with regards to Sox9 and MYCN expression; MYCN exclusive/Sox9 exclusive/intermediate population with an expression of both MYCN and Sox9. The top ∼1% of cells with the highest MYCN and Sox9 expression, respectively, and ∼1% of cells with intermediary expression were selected for the analysis in B. D, WB showing protein levels of SOX9 and MYC with actin as loading control in CHLA-01-MED and CHLA-01R-MED. SOX9 is higher in the recurrent cell line. E, The total numbers and percentages of cells with any (>0) expression of either MYC, SOX9, both, or neither in diagnostic Group 3 MBs (data from ref. 32). F, Cells from E with only SOX9 expression display a significantly higher ssGSEA score for a quiescent NSC signature as compared with cells with only MYC expression (Welch t test; P < 0.05). G, MB002 cells were transduced with lentiviral vectors encoding inducible EV, SOX9WT, or SOX9CPDmt. The transduced cell lines were treated with dox for 8 or 24 hours and analyzed for protein levels of SOX9, cMYC, and GAPDH. After the induction of SOX9, cMYC levels decreased. The decrease was more pronounced when the stabilized SOX9CPDmt was induced. H, MB002-SOX9CPDmt was treated with dox for 24 hours (for SOX9 induction) after which they were resuspended in fresh media. SOX9, cMYC, and cyclophilin B (CypB) protein were analyzed with WB at 8, 24, 48, and 72 hours after dox removal. After 72 hours, protein levels decreased but neither SOX9 nor cMYC was restored to start levels. I, Quantification by FACS of GFP-positive (+), negative (−), and intermediate cells in FACS sorted GTML-pSOX9-GFP cell line, directly after sorting, 48 hours, 1 week, and 3 weeks after sorting. The number of GFP-positive cells in the positive population quickly dropped while it slowly increased in the negative population. See also Supplementary Fig. S2.

SOX9 anticorrelates with MYC and marks quiescent cells in Group 3 patients with MB. A, Genes with higher ATAC-seq peaks in GFP-positive GTML-pSOX9-GFP cells significantly overlap with a quiescent signature (Fisher exact test; P < 0.05). B, Boxplot comparing the ssGSEA scores for a quiescent NSC signature between the groups from C, indicating that Sox9 high GTML cells appear significantly more quiescent than MYCN-high and intermediate cells (Welch t test; P < 0.05). C, Single-cell sequencing of GTML cells indicates that there exist three populations with regards to Sox9 and MYCN expression; MYCN exclusive/Sox9 exclusive/intermediate population with an expression of both MYCN and Sox9. The top ∼1% of cells with the highest MYCN and Sox9 expression, respectively, and ∼1% of cells with intermediary expression were selected for the analysis in B. D, Western blot showing protein levels of SOX9 and MYC, with actin as loading control in CHLA-01-MED and CHLA-01R-MED. SOX9 is higher in the recurrent cell line. E, The total numbers and percentages of cells with any (>0) expression of either MYC, SOX9, both, or neither in diagnostic Group 3 MBs (data from ref. 32). F, Cells from E with only SOX9 expression display a significantly higher ssGSEA score for a quiescent NSC signature as compared with cells with only MYC expression (Welch t test; P < 0.05). G, MB002 cells were transduced with lentiviral vectors encoding inducible EV, SOX9WT, or SOX9CPDmt. The transduced cell lines were treated with dox for 8 or 24 hours and analyzed for protein levels of SOX9, cMYC, and GAPDH. After the induction of SOX9, cMYC levels decreased. The decrease was more pronounced when the stabilized SOX9CPDmt was induced. H, MB002-SOX9CPDmt was treated with dox for 24 hours (for SOX9 induction), after which, they were resuspended in fresh media. SOX9, cMYC, and cyclophilin B (CypB) protein were analyzed with Western blot at 8, 24, 48, and 72 hours after dox removal. After 72 hours, protein levels decreased but neither SOX9 nor cMYC was restored to start levels. I, Quantification by FACS of GFP-positive (+), negative (−), and intermediate cells in FACS sorted GTML-pSOX9-GFP cell line, directly after sorting, 48 hours, 1 week, and 3 weeks after sorting. The number of GFP-positive cells in the positive population quickly dropped while it slowly increased in the negative population. See also Supplementary Fig. S2.