lentiviral clones expressing mbd3 shrna #2  (Addgene inc)

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic modulator inhibition overcomes temozolomide chemoresistance and antagonizes tumor recurrence of glioblastoma

doi: 10.1172/JCI127916

Figure Lengend Snippet: (A–J) pLuc-U118MG cells or pLuc-U251MG cells depleted of MBD3 by shMBD3 or shScramble lentiviral controls were intracranially injected into immunocompromised (NSG) mice. (A–D) Serial bioluminescence imaging was used to monitor tumor volume (each group, n = 5). (B and D) Tumor volume was measured every 3 or 4 days. Quantification (total flux; p/s, photons per second) of the bioluminescent signal from tumor regions in A and C. (E–J) After 4 weeks, mice were sacrificed and analyzed immunohistochemically with the indicated antibodies. Expression of CD133, CD44, CXCR4, and MBD3 (F and I) or Ki67 and nestin (G and J) was quantified using ImageJ software. Images were captured using a Zeiss confocal microscope. Representative images were selected from at least 3 different fields. Data are presented as the mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005 by 2-way ANOVA with Bonferroni’s post hoc test (B and D) or unpaired, 2-tailed Student’s t test (F, G, I, and J). Scale bars: 50 μm; and inset scale bars: 50 μm.

Article Snippet: Sequence-verified shRNA lentiviral plasmid vectors for human MBD3 and CK1A genes were subcloned into the pLKO.1 vector, and BTRCP siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Injection, Imaging, Expressing, Software, Microscopy

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic modulator inhibition overcomes temozolomide chemoresistance and antagonizes tumor recurrence of glioblastoma

doi: 10.1172/JCI127916

Figure Lengend Snippet: (A–D) ChIP-qPCR analysis of MBD3, HDAC1, HDAC2, MTA1, and acetyl–histone H3 occupancy at MBD3-binding locus in U118MG or U251MG cells treated with shScramble (n = 3) and shMBD3 (n = 3) lentiviral vectors. Immunoglobulin G (IgG) ChIP served as a negative control. Values are normalized to input control and represent the mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005 by unpaired 2-tailed Student’s t test.

Article Snippet: Sequence-verified shRNA lentiviral plasmid vectors for human MBD3 and CK1A genes were subcloned into the pLKO.1 vector, and BTRCP siRNA was purchased from Santa Cruz Biotechnology.

Techniques: Binding Assay, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: The MBD3/NuRD complex promotes H3K27 deacetylation on STAT1 promoter to inhibit STAT1 expression in GSCs. (A) The UCSC Genome Browser shows the acetylation of H3K27 on the promoter of STAT1 in GM12878 (B-lymphocyte), hESC (human embryonic stem cells), HSMM (skeletal muscle myoblasts), HUVEC (human umbilical vein endothelial cell), K562 (leukemia), NHEK (epidermal keratinocytes), and NHLF (lung fibroblasts) cells. (B and C) ChIP analyses on STAT1 promoter in GSCs/NSTCs or GSCs expressing shNT/shMBD3s. Assays were performed with the H3 antibody, and immunoprecipitates were subjected to qPCR analyses ( n = 3). (D) IB analysis of the indicated genes in 4 GSCs and matched NSTCs derived from 4 human GBM tumors. (E) Liquid chromatography-tandem MS (LC MS/MS) analysis of the purified MBD3/NuRD complex in GSC. Flag IP was performed in T387 GSC expressing Flag-MBD3. The components of NuRD complex were identified with MS. (F) IP of MBD3 was performed in T4121GSCs (left) and T387GSCs (right). The IB for CDH4, HDAC1, MBD3, and MBD2 are shown. IgG was used as an antibody control for IPs. Asterisks indicate nonspecific bands. (G) Real-time qPCR analysis of mRNA levels of MBD3, STAT1, and STAT3 in T4121GSCs or T387GSCs expressing shNT or shMBD3s ( n = 3). (H) Knockdown of MBD3 increased the expression of STAT1 in both mRNA and protein in D456 GSCs ( n = 3). (I) IB analysis of STAT1, p21, and H3K27ac in GSCs treated with SAHA for the indicated times. (J and K) T387 GSCs (J, n = 3) and T4121 GSCs (K, n = 3) were treated with the indicated dose of IFN-α/IFN-β in the absence or presence of SAHA (2 mM) for 3 d. Cell viability was assessed and normalized to the untreated control. 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. ns, not significant.

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Expressing, Derivative Assay, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Purification, Control, Knockdown

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: The MBD3/NuRD complex promotes H3K27 deacetylation on STAT1 promoter to inhibit STAT1 expression in GSCs. (A) ChIP analyses on STAT1 promoter. Assays were performed with the H3K27ac (left, n = 3) and H3K27me3 (right, n = 3) antibodies, and immunoprecipitates were subjected to qPCR analyses. (B) ChIP analysis with MBD3 antibody showing the enrichment of MBD3 at the promoter of STAT1 (around primer 6) in T4121GSCs and T387GSCs. Schematic showing the ChIP primer location with respect to the TSS of the STAT1 promoter (top). (C and D) ChIP analysis on the promoter of STAT1 in T4121GSCs ( n = 3) and T387GSCs ( n = 3) expressing shNT or two independent shMBD3s. Assays were performed with the indicated antibodies, and immunoprecipitates were subjected to qPCR analyses (primer 6). (E) IB analysis of STAT1, STAT3, and MBD3 in T387GSCs and T4121GSCs expressing shNT or two independent shMBD3s. (F) ChIP analyses on STAT1 promoter in GSCs and matched NSTCs. Assays were performed with the HDAC1 (left, n = 3) and CHD4 (right, n = 3) antibodies, and immunoprecipitates were subjected to qPCR analyses (primer 6). (G) Proposed model for MBD3/NuRD-mediated regulation of STAT1 transcription. In GSCs, MBD3 is highly expressed and binds to STAT1 promoter, recruits the NuRD complex (including CHD4 and HDAC1) to suppress STAT1 expression by H3K27 deacetylation. Loss of MBD3 disassembles the NuRD complex, increases H3K27 acetylation, and promotes STAT1 transcription. 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.

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: MBD3 is preferentially expressed in GSCs. (A) IB analysis of MBD3, MBD2, SOX2, and GFAP in GSCs and matched NSTCs derived from five human GBM tumors. (B) IB analysis of MBD3, MBD2, SOX2, and GFAP during GSC differentiation. (C) IB analysis of MBD3, STAT1, SOX2, and Olig2 in GSCs and NHAs. (D) Co-IF staining of MBD3 (green) and SOX2/Olig2 (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue). (E) IHC staining of MBD3 in brain tumor tissue microarray. Section was counterstained with hematoxylin (left). Box plot of histoscore of MBD3 (right). Normal brain tissue ( n = 5), low-grade gliomas (I–II, n = 15), and high-grade gliomas (III–IV, n = 39). One-way ANOVA; *, P < 0.05. (F) IHC staining of MBD3 (left) and STAT1 (right) in serial sections of human GBM specimens. Sections were counterstained with hematoxylin. (G) Co-IF staining of STAT1 (green) and MBD3 (red) in human GBM specimen and mouse GBM orthotopic xenograft. Nuclei were counterstained with Hoechst (blue).

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Derivative Assay, Staining, Immunohistochemistry, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: MBD3 is preferentially expressed in GSCs. (A) Co-IF staining of MBD3 (green) and SOX2, NESTIN (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue). (B and C) Co-IF staining of MBD3 (green) and SOX2, Olig2, NESTIN (red) in mouse GBM xenografts. Nuclei were counterstained with Hoechst (blue). (D) Pairwise correlation analysis of the indicated genes in TCGA GBM database. Pearson correlation coefficient (r) value and P value are shown ( n = 538). (E–H) IHC staining of SOX2, MBD3, and STAT1 in the serial sections of human glioma tissue microarrays. Sections were counterstained with hematoxylin (E, F, and H). IHC score of MBD3 in brain tumor tissue microarray (E). Boxplot (G, left) and correlation analysis (G, right; n = 35) of histoscores of the tissue microarray stained for indicated proteins are shown. Low-grade gliomas (I–II, n = 13) and high-grade gliomas (III–IV, n = 42). SOX2 + cells were quantified to imply the fraction of GSCs in tumor (G; low GSCs, n = 21; high GSCs, n = 19). The scale bar represents 50 μm (F). *, P < 0.05; ***, P < 0.001, as assayed by unpaired Student’s t test. (I) Co-IF staining of STAT1 (green) and MBD3 (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue).

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Staining, Immunohistochemistry, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: 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.

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Inhibition, Expressing, Luciferase, Reporter Assay, Knockdown, Activation Assay, Immunohistochemistry, Derivative Assay, Staining, shRNA, Control

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Depletion of MBD3 upregulates IFN signaling and inhibits GSC growth. (A) Real-time qPCR analysis of mRNA levels of MCM10, POLA1, CDK4, and CDC45 in T387GSCs expressing shNT or shMBD3 ( n = 3). (B) CDKN1A promoter (WWP-Luc) luciferase reporter assay showed that MBD3 depletion had no effect on the reporter with STAT1 binding site mutation. Binding sites of STAT1 on CDKN1A promoter was mutated from 5′-TTCCCGGAA-3′ to 5′-AAGCTTGAA-3′ ( n = 3). (C) Knockdown of MBD3 inhibited GSC proliferation assessed by EdU incorporation assay and Ki67 staining in T4121 GSCs expressing shNT or shMBD3. Representative images are shown (left). The percentage of EdU + or Ki67 + cells was quantified (right; n = 3). (D) IB analysis showed the knockdown of MBD3 with two different shRNAs in T4121GSCs and T387GSCs. (E) Knockdown of MBD3 inhibited D456 GSC sphere formation. (F) Cell viability of T387 NSTCs expressing shNT or shMBD3 ( n = 3). (G) Representative images of cross sections (H&E stain) of mouse brains (nu/nu) 38 d after transplantation with T387 GSC expressing shNT, shMBD3#1, or shMBD3#2. (H) T4121 GSCs transduced with Tet-on-shMBD3 were treated with Dox (100 ng/ml) or vehicle control. IB analysis showed the knockdown of MBD3 in T4121 GSCs (left). Inducible knockdown of MBD3 inhibited T4121 GSCs tumorsphere formation (middle) and cell viability (right; n = 3). (I) IF staining of MBD3 (red) in xenograft tissues to assess the efficiency of MBD3 knockdown in vivo in , respectively. (J) IF staining of SOX2 or GFAP (red) in xenografts of T4121 GSCs (Dox-shMBD3) implanting mice (nu/nu) treated with or without Dox. Quantification of SOX2 or GFAP percentage are shown (right, n = 5). (K) Kaplan–Meier survival analysis of patients with high ( n = 76) and low ( n = 79) expression of MBD3 in Gravendeel GBM dataset. Log-rank test. For A–J, 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.

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Expressing, Luciferase, Reporter Assay, Binding Assay, Mutagenesis, Knockdown, Staining, Transplantation Assay, Transduction, Control, In Vivo

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Highly expressed MBD3 promotes GSC malignant progression . (A) GSCs expressing shNT or shMBD3s were transplanted into brains of nude mice (5 × 10 4 cells/mouse). Kaplan–Meier survival curves of mice implanted with T4121 GSCs (shNT, n = 7; shMBD3#1, n = 6; shMBD3#2, n = 7) or T387 GSCs (shNT, n = 6; shMBD3#1, n = 8; shMBD3#2, n = 7) are shown. Log-rank test. (B) T4121 GSCs expressing shNT or shMBD3 were transplanted into brains of nude mice in a limiting dilution manner (2 × 10 5 or 2 × 10 4 cells/mouse, n = 9 or n = 8, respectively). Kaplan–Meier survival plots are shown. Log-rank test. (C–F) Luciferase-labeled T4121GSCs were transduced with the Tet-on-inducible shMBD3 and then transplanted into the brains of nude mice (2 × 10 4 cells/mouse). Mice were treated with vehicle control or Dox (2 mg/ml in drinking water) to induce expression of shMBD3 from day 0 (C and E) or day 14 (D and F). GBM xenografts were tracked by bioluminescence, and the representative images from animals at the indicated time are shown (C and D, left). Bioluminescent quantification indicated that induced knockdown of MBD3 inhibited GSC tumor initiation and growth (C and D, right). Kaplan–Meier survival plots of mice are shown (E, shNT, n = 8; shMBD3, n = 10; F, n = 7 for each group). Unpaired Student’s t test for C and D. Log-rank test for E and F. (G) Co-IF staining of Ki67 and MBD3 in GBM xenografts derived from T387 GSCs expressing shNT or shMBD3 (left). The percentage of Ki67 + cells was quantified (right, n = 3). Data are represented as mean ± SD (unpaired Student’s t test). (H) Kaplan–Meier survival analysis of patients with high ( n = 93) and low expression ( n = 88) of MBD3 in REMBRANDT GBM dataset. Log-rank test. (I) Knockout of STAT1 rescued the inhibition of MBD3 depletion on GSC viability and tumor initiation. IB of WT and STAT1 KO GSCs transduced with shNT or shMBD3 (left). Cell viability was assessed with GSCs as indicated (middle, n = 3, unpaired Student’s t test). The indicated GSCs were transplanted into brains of nude mice (2 × 10 4 cells/mouse). Kaplan–Meier survival curves of mice are shown ( n = 6 for each group). Log-rank test. Data are represented as mean ± SD (G and I) or mean ± SEM (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001. nu/nu nude mice were used in the animal experiments.

Article Snippet: Lentiviral clones expressing MBD3 shRNA (#1, 5′-CAA GAT GCT GAT GAG CAA GAT-3′; #2, 5′-CCT GTG CAA AGC CTT CAT GGT-3′) were prepared according to the modified protocols from Addgene ( https://www.addgene.org/tools//protocols/plko/ ).

Techniques: Expressing, Luciferase, Labeling, Transduction, Control, Knockdown, Staining, Derivative Assay, Knock-Out, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: The MBD3/NuRD complex promotes H3K27 deacetylation on STAT1 promoter to inhibit STAT1 expression in GSCs. (A) The UCSC Genome Browser shows the acetylation of H3K27 on the promoter of STAT1 in GM12878 (B-lymphocyte), hESC (human embryonic stem cells), HSMM (skeletal muscle myoblasts), HUVEC (human umbilical vein endothelial cell), K562 (leukemia), NHEK (epidermal keratinocytes), and NHLF (lung fibroblasts) cells. (B and C) ChIP analyses on STAT1 promoter in GSCs/NSTCs or GSCs expressing shNT/shMBD3s. Assays were performed with the H3 antibody, and immunoprecipitates were subjected to qPCR analyses ( n = 3). (D) IB analysis of the indicated genes in 4 GSCs and matched NSTCs derived from 4 human GBM tumors. (E) Liquid chromatography-tandem MS (LC MS/MS) analysis of the purified MBD3/NuRD complex in GSC. Flag IP was performed in T387 GSC expressing Flag-MBD3. The components of NuRD complex were identified with MS. (F) IP of MBD3 was performed in T4121GSCs (left) and T387GSCs (right). The IB for CDH4, HDAC1, MBD3, and MBD2 are shown. IgG was used as an antibody control for IPs. Asterisks indicate nonspecific bands. (G) Real-time qPCR analysis of mRNA levels of MBD3, STAT1, and STAT3 in T4121GSCs or T387GSCs expressing shNT or shMBD3s ( n = 3). (H) Knockdown of MBD3 increased the expression of STAT1 in both mRNA and protein in D456 GSCs ( n = 3). (I) IB analysis of STAT1, p21, and H3K27ac in GSCs treated with SAHA for the indicated times. (J and K) T387 GSCs (J, n = 3) and T4121 GSCs (K, n = 3) were treated with the indicated dose of IFN-α/IFN-β in the absence or presence of SAHA (2 mM) for 3 d. Cell viability was assessed and normalized to the untreated control. 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. ns, not significant.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing, Derivative Assay, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Purification

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: The MBD3/NuRD complex promotes H3K27 deacetylation on STAT1 promoter to inhibit STAT1 expression in GSCs. (A) ChIP analyses on STAT1 promoter. Assays were performed with the H3K27ac (left, n = 3) and H3K27me3 (right, n = 3) antibodies, and immunoprecipitates were subjected to qPCR analyses. (B) ChIP analysis with MBD3 antibody showing the enrichment of MBD3 at the promoter of STAT1 (around primer 6) in T4121GSCs and T387GSCs. Schematic showing the ChIP primer location with respect to the TSS of the STAT1 promoter (top). (C and D) ChIP analysis on the promoter of STAT1 in T4121GSCs ( n = 3) and T387GSCs ( n = 3) expressing shNT or two independent shMBD3s. Assays were performed with the indicated antibodies, and immunoprecipitates were subjected to qPCR analyses (primer 6). (E) IB analysis of STAT1, STAT3, and MBD3 in T387GSCs and T4121GSCs expressing shNT or two independent shMBD3s. (F) ChIP analyses on STAT1 promoter in GSCs and matched NSTCs. Assays were performed with the HDAC1 (left, n = 3) and CHD4 (right, n = 3) antibodies, and immunoprecipitates were subjected to qPCR analyses (primer 6). (G) Proposed model for MBD3/NuRD-mediated regulation of STAT1 transcription. In GSCs, MBD3 is highly expressed and binds to STAT1 promoter, recruits the NuRD complex (including CHD4 and HDAC1) to suppress STAT1 expression by H3K27 deacetylation. Loss of MBD3 disassembles the NuRD complex, increases H3K27 acetylation, and promotes STAT1 transcription. 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.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: MBD3 is preferentially expressed in GSCs. (A) IB analysis of MBD3, MBD2, SOX2, and GFAP in GSCs and matched NSTCs derived from five human GBM tumors. (B) IB analysis of MBD3, MBD2, SOX2, and GFAP during GSC differentiation. (C) IB analysis of MBD3, STAT1, SOX2, and Olig2 in GSCs and NHAs. (D) Co-IF staining of MBD3 (green) and SOX2/Olig2 (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue). (E) IHC staining of MBD3 in brain tumor tissue microarray. Section was counterstained with hematoxylin (left). Box plot of histoscore of MBD3 (right). Normal brain tissue ( n = 5), low-grade gliomas (I–II, n = 15), and high-grade gliomas (III–IV, n = 39). One-way ANOVA; *, P < 0.05. (F) IHC staining of MBD3 (left) and STAT1 (right) in serial sections of human GBM specimens. Sections were counterstained with hematoxylin. (G) Co-IF staining of STAT1 (green) and MBD3 (red) in human GBM specimen and mouse GBM orthotopic xenograft. Nuclei were counterstained with Hoechst (blue).

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Derivative Assay, Staining, Immunohistochemistry, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: MBD3 is preferentially expressed in GSCs. (A) Co-IF staining of MBD3 (green) and SOX2, NESTIN (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue). (B and C) Co-IF staining of MBD3 (green) and SOX2, Olig2, NESTIN (red) in mouse GBM xenografts. Nuclei were counterstained with Hoechst (blue). (D) Pairwise correlation analysis of the indicated genes in TCGA GBM database. Pearson correlation coefficient (r) value and P value are shown ( n = 538). (E–H) IHC staining of SOX2, MBD3, and STAT1 in the serial sections of human glioma tissue microarrays. Sections were counterstained with hematoxylin (E, F, and H). IHC score of MBD3 in brain tumor tissue microarray (E). Boxplot (G, left) and correlation analysis (G, right; n = 35) of histoscores of the tissue microarray stained for indicated proteins are shown. Low-grade gliomas (I–II, n = 13) and high-grade gliomas (III–IV, n = 42). SOX2 + cells were quantified to imply the fraction of GSCs in tumor (G; low GSCs, n = 21; high GSCs, n = 19). The scale bar represents 50 μm (F). *, P < 0.05; ***, P < 0.001, as assayed by unpaired Student’s t test. (I) Co-IF staining of STAT1 (green) and MBD3 (red) in human GBM specimens. Nuclei were counterstained with Hoechst (blue).

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Staining, Immunohistochemistry, Microarray

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: 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.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Inhibition, Expressing, Luciferase, Reporter Assay, Activation Assay, Immunohistochemistry, Derivative Assay, Staining, shRNA

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Depletion of MBD3 upregulates IFN signaling and inhibits GSC growth. (A) Real-time qPCR analysis of mRNA levels of MCM10, POLA1, CDK4, and CDC45 in T387GSCs expressing shNT or shMBD3 ( n = 3). (B) CDKN1A promoter (WWP-Luc) luciferase reporter assay showed that MBD3 depletion had no effect on the reporter with STAT1 binding site mutation. Binding sites of STAT1 on CDKN1A promoter was mutated from 5′-TTCCCGGAA-3′ to 5′-AAGCTTGAA-3′ ( n = 3). (C) Knockdown of MBD3 inhibited GSC proliferation assessed by EdU incorporation assay and Ki67 staining in T4121 GSCs expressing shNT or shMBD3. Representative images are shown (left). The percentage of EdU + or Ki67 + cells was quantified (right; n = 3). (D) IB analysis showed the knockdown of MBD3 with two different shRNAs in T4121GSCs and T387GSCs. (E) Knockdown of MBD3 inhibited D456 GSC sphere formation. (F) Cell viability of T387 NSTCs expressing shNT or shMBD3 ( n = 3). (G) Representative images of cross sections (H&E stain) of mouse brains (nu/nu) 38 d after transplantation with T387 GSC expressing shNT, shMBD3#1, or shMBD3#2. (H) T4121 GSCs transduced with Tet-on-shMBD3 were treated with Dox (100 ng/ml) or vehicle control. IB analysis showed the knockdown of MBD3 in T4121 GSCs (left). Inducible knockdown of MBD3 inhibited T4121 GSCs tumorsphere formation (middle) and cell viability (right; n = 3). (I) IF staining of MBD3 (red) in xenograft tissues to assess the efficiency of MBD3 knockdown in vivo in , respectively. (J) IF staining of SOX2 or GFAP (red) in xenografts of T4121 GSCs (Dox-shMBD3) implanting mice (nu/nu) treated with or without Dox. Quantification of SOX2 or GFAP percentage are shown (right, n = 5). (K) Kaplan–Meier survival analysis of patients with high ( n = 76) and low ( n = 79) expression of MBD3 in Gravendeel GBM dataset. Log-rank test. For A–J, 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.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing, Luciferase, Reporter Assay, Binding Assay, Mutagenesis, Staining, Transplantation Assay, Transduction, In Vivo

Journal: The Journal of Experimental Medicine

Article Title: Glioma stem-like cells evade interferon suppression through MBD3/NuRD complex–mediated STAT1 downregulation

doi: 10.1084/jem.20191340

Figure Lengend Snippet: Highly expressed MBD3 promotes GSC malignant progression . (A) GSCs expressing shNT or shMBD3s were transplanted into brains of nude mice (5 × 10 4 cells/mouse). Kaplan–Meier survival curves of mice implanted with T4121 GSCs (shNT, n = 7; shMBD3#1, n = 6; shMBD3#2, n = 7) or T387 GSCs (shNT, n = 6; shMBD3#1, n = 8; shMBD3#2, n = 7) are shown. Log-rank test. (B) T4121 GSCs expressing shNT or shMBD3 were transplanted into brains of nude mice in a limiting dilution manner (2 × 10 5 or 2 × 10 4 cells/mouse, n = 9 or n = 8, respectively). Kaplan–Meier survival plots are shown. Log-rank test. (C–F) Luciferase-labeled T4121GSCs were transduced with the Tet-on-inducible shMBD3 and then transplanted into the brains of nude mice (2 × 10 4 cells/mouse). Mice were treated with vehicle control or Dox (2 mg/ml in drinking water) to induce expression of shMBD3 from day 0 (C and E) or day 14 (D and F). GBM xenografts were tracked by bioluminescence, and the representative images from animals at the indicated time are shown (C and D, left). Bioluminescent quantification indicated that induced knockdown of MBD3 inhibited GSC tumor initiation and growth (C and D, right). Kaplan–Meier survival plots of mice are shown (E, shNT, n = 8; shMBD3, n = 10; F, n = 7 for each group). Unpaired Student’s t test for C and D. Log-rank test for E and F. (G) Co-IF staining of Ki67 and MBD3 in GBM xenografts derived from T387 GSCs expressing shNT or shMBD3 (left). The percentage of Ki67 + cells was quantified (right, n = 3). Data are represented as mean ± SD (unpaired Student’s t test). (H) Kaplan–Meier survival analysis of patients with high ( n = 93) and low expression ( n = 88) of MBD3 in REMBRANDT GBM dataset. Log-rank test. (I) Knockout of STAT1 rescued the inhibition of MBD3 depletion on GSC viability and tumor initiation. IB of WT and STAT1 KO GSCs transduced with shNT or shMBD3 (left). Cell viability was assessed with GSCs as indicated (middle, n = 3, unpaired Student’s t test). The indicated GSCs were transplanted into brains of nude mice (2 × 10 4 cells/mouse). Kaplan–Meier survival curves of mice are shown ( n = 6 for each group). Log-rank test. Data are represented as mean ± SD (G and I) or mean ± SEM (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001. nu/nu nude mice were used in the animal experiments.

Article Snippet: The inducible shMBD3 construct was constructed by insertion of MBD3 shRNA (5′-CAA​GAT​GCT​GAT​GAG​CAA​GAT-3′) into Tet-pLKO-puro (Addgene).

Techniques: Expressing, Luciferase, Labeling, Transduction, Staining, Derivative Assay, Knock-Out, Inhibition