Fig. 4: Inhibiting OLIG2 enriches for less proliferative and a more potent sphere-forming stem cell population.

a Structure of the OLIG2 inhibitor, CT-179⁶⁷. b, c Immunocytochemistry of Ptch1^+/−;Trp53^−/− mouse tumour cells (b), quantification of EdU incorporation in OLIG2⁺ cells after a 1-hour pulse (c); data shown are representative of three independent experiments; error bars denote mean ± SEM; two-tailed unpaired t-test; ***p < 0.001; p = 0.0001 (c). d Proportion of Ptch1^+/−;Trp53^−/− mouse tumour cells in each phase of the cell cycle as measured through FACS analysis of propidium iodide (PI) staining and analysed using FlowJo software; data shown are representative of three independent experiments; error bars denote mean ± SEM; two-tailed unpaired t-test; *p < 0.05; p = 0.0143 (G0/G1), p = 0.0157 (S), p = 0.0210 (G2/M). e Percentage confluence of Ptch1^+/−;Trp53^−/− mouse tumour cells transfected with a doxycycline (DOX)-inducible OLIG2 overexpression (O/E) construct or empty vector control upon treatment with CT-179 or vehicle for 10 days; data shown are representative of three independent experiments; error bars denote mean ± SEM; two-tailed unpaired t-test; *p < 0.05; p = 0.0404 (O/E vector control vs. OLIG2 O/E), p = 0.0484 (OLIG2 O/E vs. OLIG2 O/E + CT-179). f–h Immunocytochemistry of Ptch1^+/−;Trp53^−/− mouse tumour cells treated with IC₉₀ of CT-179 (276.1nM) (f), quantification of SOX2⁺ cells (g), quantification of the fluorescence intensity of DAPI, SOX2 and OLIG2 measured using ImageJ software (h); data shown in are representative of three independent experiments; scale bar: 80µM (f); g, h: error bars denote mean ± SEM; two-tailed unpaired t-test; ns p ≥ 0.05, **p < 0.01, ***p < 0.001; p = 0.0001 (SOX2) (g); p = 0.4551 (DAPI), p = 0.0003 (SOX2), p = 0.0035 (OLIG2) (h). i, j Secondary limiting dilution analysis (LDA) performed on Ptch1^+/−;Trp53^−/− mouse tumour cells pre-treated with vehicle, IC₁₀ (129.7 nM), IC₅₀ (189.2 nM) or IC₉₀ (276.1nM) dose of CT-179 for 24 hours; data show the percentage of sphere-forming capacity (i) and a quantification of the spheres size (j); data shown are representative of three independent experiments; line at the median; two-tailed unpaired t-test; ns p ≥ 0.05, *p < 0.05, ****p < 0.0001; p = 0.0724 (IC₁₀ vs. vehicle), p = 0.0259 (IC₅₀ vs. vehicle), p = 0.0259 (IC₉₀ vs. vehicle) (i); p = 0.3791 (IC₁₀ vs. vehicle), p < 0.0001 (IC₅₀ vs. vehicle), p < 0.0001 (IC₉₀ vs. vehicle) (j). k, l Cell confluence measured using high-throughput live-cell imaging of Ptch1^+/−;Trp53^−/− mouse tumour cells pre-treated with vehicle or IC₅₀ (189.2 nM) CT-179 for 24 hours, immediately followed by a dose-response assay to AraC (k) and Vismodegib (GDC) (l). Source data are provided as a Source Data file.