Figure 2. KLHDC8A is necessary for GSC maintenance.

(A and B) Cell viability was measured by CellTiter-Glo assay in paired GSC23, GSC3028, and differentiated counterparts (DGC23 and DGC3028) over a 6-day time course following KLHDC8A knockdown. n = 4. Quantitative data from 4 technical replicates are shown as mean ± SD. Statistical analysis was performed using 2-way ANOVA with Dunnett’s multiple comparisons. (C and D) The knockdown efficiency of KLHDC8A was measured by qPCR in GSCs (C) and DGCs (D). n = 4. Quantitative data from 4 independent experiments are shown as mean ± SD. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons. (E) In vitro ELDA in GSC23 and GSC3028 following knockdown of KLHDC8A. 24 wells were quantified for each condition. Statistical analysis was performed using χ² test for pairwise differences. (F) The knockdown efficiency of KLHDC8A was measured by qPCR in GSC3028 and GSC23. n = 4. Quantitative data from 4 independent experiments are shown as mean ± SD. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons. (G) Immunoblot showing protein levels of PARP and cleaved PARP in GSC387 and GSC23 following KLHDC8A knockdown. β-Actin was used as the loading control. (H) Annexin V staining of GSC23 and GSC387 was performed following knockdown of KLHDC8A. (I) Quantification of Annexin V staining in GSC387 and GSC23. n = 3. Quantitative data from 3 technical replicates are shown as mean ± SD. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons. (J) Protein levels of OLIG2 and SOX2 following KLHDC8A knockdown were measured by immunoblot. β-Actin was used as the loading control. (K) Protein levels of SOX2 following KLHDC8A knockdown in mesenchymal and proneural subtypes of GSCs were measured by immunoblot. β-Actin was used as the loading control. **P < 0.01, ****P < 0.0001.