Fig 1: Radiosensitization in DNMT3B knockdown cells.(A) The DNMT3B knockdown increased radiosensitivity of HeLa cells. The cells were treated with shRNA of DNMT3B and the mRNA level and protein level of DNMT3B was detected by qRT-PCR (a) and western blot (a, inserted), respectively. The effect of DNMT3B knockdown with shRNA on the radiosensitivity of HeLa cells was analyzed by colony formation assay (b). The enhancement ratio at 10% survival was 1.2 (c-e). The knockdown levels of DNMT3B after transfection of DNMT3B specific siRNA at the mRNA (c) and protein (c, inserted) levels. The effect of DNMT3B knockdown with siRNA on the radiosensitivity of HeLa cells was analyzed by colony formation assay (e). The enhancement ratio at 10% survival was 1.3. (B and C) DNMT3B RNAi radiosensitized in A549 (B) and HCT116 (C) cell lines. The mRNA and protein levels of DNMT3B in A549 transfected with shRNA for DNMT3B (Ba with an inserted panel) and HCT116 transfected siRNA for DNMT3B (Ca with an inserted panel). Error bars: SE. The colony formation analysis was carried out (Bb and Cb). Error bars: SE. The enhancement ratio at 10% survival was 1.2 for A549 (Bb) and 1.4 for HCT116 (Cb). Asterisks show P < 0.05. The mRNA level of DNMT3B was normalized with that of GUSB (Aa, Ac, Ba and Ca).
Fig 2: Knockdown of DNMT3B enhances radiosensitivity in tumor xenograft model.An overview of the xenograft experimental procedure (A). The temporal development of the tumor masses in animals injected with control and DNMT3B knockdown A549 cells (B). Error bars: SE. Asterisks show P < 0.05. The tumor weights at day 38 showing that knockdown of DNMT3B increased the susceptibility of the tumors to γ-irradiation. Bars show the mean tumor weight. Asterisks show P < 0.05 (C).
Fig 3: Knockdown of DNMT3B radiosensitizes through impairment of H2AX regulation after DNA damage.(A) H2AX overexpression suppressed radiosensitization induced by DNMT3B RNAi in HeLa cells. MTT assay was performed 4 days after γ-irradiation. Relative mRNA expression levels of DNMT3B and H2AX in indicated conditions (a). The expression level was normalized to that of GUSB. Error bars: SE. Relative growth levels of HeLa cells in each transfected condition (b). Error bars: SE. (B) Interaction between DNMT3B and HP1β or H2AX observed 1 hr after 8 Gy irradiation. Confirmation of DNMT3B-FLAG overexpression at mRNA level (a) and protein level (b). The number of PLA foci between DNMT3B and HP1β was decreased after irradiation (c), whereas that of DNMT3B and H2AX was increased after irradiation (d). Scale bar: 10 μm.
Fig 4: Knockdown of DNMT3B disrupts γ-irradiation-induced formation of HP1β foci.(A and B) Formation of HP1β foci in control and DNMT3B knockdown cells 1 h after irradiation (8 Gy). (A) Immunostaining data. Arrows: HP1β foci merged with H3K9me2/3. Arrows heads: HP1β foci not merged with H3K9me2/3. (B) Quantitative data. HP1β foci were counted at least in 50 cells. Significant differences between the number of foci in non-irradiated and irradiated cells were determined. Error bars: SE. Asterisks show P < 0.05. Scale bar: 10 μm. N.S.: P ≧ 0.05.
Fig 5: Comprehensive analysis identified radiosensitizing targets including DNMT3B.(A and B) The results of a comprehensive negative screening analysis of radiosensitivity-related genes in T-REx HeLa cells transfected with a shRNA library. (C) Analysis of a functional network cluster around the genes encoding phosphatase and tensin homolog (PTEN) and glycogen synthase kinase 3β (GSK3β) (upper panel). The numbers shown in blue indicate genes whose knockdown induced radiosensitization (lower panel). (D) Confirmation of RNAi-mediated knockdown of CUL1 (a) and FBXW7 (b) expression in T-REx HeLa cells. Error bars: SE. Asterisks show P < 0.05. (E) Radiosensitization induced by CUL1 or FBXW7 knockdown in T-REx HeLa cells analyzed by colony formation assay. Mean + SE. Asterisks show P < 0.05. (F) Cluster analysis and validation of novel candidate genes. A functional network cluster of the target genes around PARP-1 (left panel). The targets whose downregulation induced radiosensitization and radioresistance are shown in blue and red, respectively (right panel).
Supplier Page from DNASU for DNMT3B (Homo sapiens) in pENTR223.1 (Gateway donor/master vector)