Fig 1: 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).
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: 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 4: Knockdown of DNMT3B reduces the formation of γH2AX foci after irradiation.(A and B) The formation of γH2AX foci 1 h and 7 h after exposure of control and DNMT3B knockdown cells to γ-irradiation (A) or carbon-ion beam irradiation (B). Failure of quick and robust γH2AX induction after γ-irradiation in DNMT3B knockdown cells (Aa); One hour after γ-irradiation, DNMT3B knockdown cells exhibited γH2AX foci with low intensity compared to that in NC cells. On the other hand, 7 h after irradiation, γH2AX signals in DNMT3B knockdown cells became more intensive compared to NC cells (Ab and Ac). Green square regions in Aa were enlarged in Ab. The γH2AX foci were counted at least in 140 cells, respectively. Asterisks show P < 0.05 (X2-test). Western blot analysis showed that carbon-ion beam irradiation also failed to the quick activation of γH2AX in DNMT3B knockdown cells (B). (C) H2AX protein levels in control and DNMT3B knockdown cells exposed to γ-irradiation. Scale bar: 20 μm. (D) H2AX mRNA levels in control and DNMT3B knockdown cells after 8 Gy γ-irradiation. The expression level of H2AX was normalized to that of GUSB. Error bars: SE.
Fig 5: Knockdown of DNMT3B activates the P53-dependent pathway after γ-irradiation of HeLa cells.(A) The G1, G2/M and sub-G1 populations of DNMT3B knockdown cells 8 h and 24 h after irradiation (8 Gy). Error bars: SE. Asterisks show P < 0.05. (B) The G1, G2/M and sub-G1 populations of synchronized DNMT3B knockdown cells 7 h after irradiation. The upper panel shows the scheme of synchronization experiment. Error bars: SE. Asterisks show P < 0.05. (C) The activation of P53 pathway genes in irradiated DNMT3B knockdown cells. The expression level was normalized to that of GUSB. Error bars: SE. (D) A model for DNMT3B functions in IR response. DNMT3B interacts with the DSB sensor proteins, H2AX and HP1β and protects cells from irradiation damage through regulation of damage associated responses.
Supplier Page from DNASU for DNMT3B (Homo sapiens) in pENTR223.1 (Gateway donor/master vector)