Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2020 Mar 17;217(5):e20191340. doi: 10.1084/jem.20191340

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2020 Zhan et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure S5. — 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 Fig. 7 (A–D), 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.