Depletion of MBD3 leads to upregulation of IFN signaling and growth inhibition in GSCs. (A) Overrepresented gene ontology terms among upregulated gene sets (left) and downregulated gene sets (right) in shMBD3-GSCs compared with the shNT-GSCs. (B) Gene set enrichment analysis shows the enrichment of gene sets positive related to immune response (left) and negative related to cell cycle process (right) in shMBD3-GSCs compared with the shNT-GSCs. (C) Heatmap representation of upregulated genes involved in IFN response (left) and downregulated genes involved in cell cycle process (right) in shMBD3-GSCs compared with the shNT-GSCs. (D) Real-time qPCR analysis of mRNA level of IRGs in T387 or T4121 GSCs expressing shNT or shMBD3 (n = 3). (E) Real-time qPCR (left) and IB (right) analysis of p21 expression in T387 or T4121 GSCs expressing shNT or shMBD3 (n = 3). (F) p21 promoter (WWP-Luc) luciferase reporter assay showed that knockdown of MBD3 induced the transcription activation of p21 in GSCs (n = 3). (G) IHC staining of p21 in GBM xenografts derived from T387 GSCs expressing shNT or shMBD3 (left). The percentage of p21⁺ cells was quantified (right; n = 3). (H) Knockdown of MBD3 impaired GSC proliferation assessed by EdU incorporation assay and Ki67 staining in T387 GSCs. Representative images are shown (left). The percentage of EdU⁺ or Ki67⁺ cells was quantified (right; n = 3). (I and J) Knockdown of MBD3 with two shRNA sequences inhibited GSC sphere formation (I) and cell viability (J; n = 3). (K) Knockdown of MBD3 had no obvious effect on cell viability of NHA (n = 3). (L) T4121 GSCs expressing shNT or shMBD3 were treated with indicated dose of IFN-α or IFN-β for 3 d, and cell viability was assessed and normalized to the untreated control (n = 3). Data are represented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, as assayed by unpaired Student’s t test or Welch’s t test.