Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 loss confers sensitivity to PARP inhibition. ( A–C ) Clonogenic survival assay of Du145, MIA-PaCa2 and U2OS cells transfected either with control siRNA (siCTR) or with a pool of four siRNAs targeting ID3 (siID3) and treated with the indicated dose of olaparib. ( D ) Clonogenic survival assay of WT and ID3-KO cells treated with the indicated doses of olaparib. ( E , F ) Clonogenic survival assay of various prostate cancer and pancreatic cancer cell lines expressing different ID3 levels treated with the indicated dose of olaparib. ( G ) Clonogenic survival assay of WT and ID3-KO U2OS cells transfected with siRECQL and treated with the indicated dose of olaparib, lower panel shows representative colony formation images. ( H ) Clonogenic survival assay of Du145 cells with single or double depletion of ID3 and RECQL and treated with the indicated dose of olaparib, lower panel shows representative colony formation images. All clonogenic survival assays are presented as mean ± SEM of three independent experiments, Student t test was performed. ( I ) Model of the multiple roles of ID3 in HR. Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Inhibition, Clonogenic Cell Survival Assay, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 interacts with DNA repair proteins after IR. ID3-KO cells expressing Flag-tagged ID3 were irradiated with 10 Gy and harvested at 15 or 60 min after irradiation. ( A ) Venn diagram showing the mass spectrometry-identified interaction peptides among unirradiated (UT) and irradiated samples after 15 and 60 min. ( B ) Pathway enrichment analysis using the interaction proteins 15 min after irradiation. Analysis was done using Metascape resources (Ref.# 71). ( C ) Pathway enrichment analysis using the interaction proteins 1h after irradiation. Analysis was done using Metascape resources (Ref.# 71). ( D ) Volcano plot showing identified ID3 interaction candidates, identified proteins are highlighted in red. Y-axis represents t -test statistics of log-10 LFQ intensities. ( E ) IP of Flag-ID3 using Flag-beads followed by Western blot to validate co-IP of the identified DNA repair proteins interacting with ID3 (Representative Western blot, left panel) and a bar plot for the quantification of three independent Western blots (right panel, mean ± SD). ( F , G ) Western blot to confirm interaction of endogenous ID3 to RAD50, NBS1 and RECQL (left panels) and bar plot showing the quantification of three independent Western blots (right panels, mean ± SD). ( H ) Western blot of the co-IP of RAD50, NBS1 and RECQL to ID3 after treatment either with DMSO or 10 μM ATMi prior to irradiation (representative Western blot, left panel) and a bar plot of the quantification of three independent Western blots (right panel, mean ± SD). (I) Western blot of the co-IP of RAD50, NBS1 and RECQL to WT-ID3, phospho-dead ID3 (S65A) and phospho-memic ID3 (S65E). ( J ) Western blot of the co-IP of RAD50, NBS1 and RECQL to endogenous ID3 upon knockdown of MDC1 prior to irradiation (representative Western blot, left panel) and a bar plot for the quantification of three independent Western blots (right panel, mean ± SD). Student's t test was used. Statistical significance is presented as: * P < 0.05 , ** P < 0.01 , ***P < 0.001 , ****P < 0.0001, ns = not significant.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Expressing, Irradiation, Mass Spectrometry, Western Blot, Co-Immunoprecipitation Assay

Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 is required for cell survival and DSB repair. ( A–D ) Clonogenic survival assay of Du145 and U2OS cells transfected either with control siRNA (siCTR) or a pool of four siRNAs targeting ID3 (siID3) and treated with the indicated dose of ionizing radiation or concentrations of cisplatin. n = 3 independent experiments;data are presented as mean ± SEM, one-way ANOVA with Bonferroni's multiple comparison test. ( E , F ) Representative micrographs and quantification of IR-induced γH2AX foci in Du145 and U2OS cells, ca. 500 cells were counted at indicated time points. n = 3 independent experiments; data are presented as mean ± SEM, one-way ANOVA with Bonferroni's multiple comparison test. ( G ) Clonogenic survival assay of prostate cancer cell lines with different ID3 expression levels treated with the indicated doses of ionizing radiation. n = 3 independent experiments; data are presented as mean ± SEM, One-Way ANOVA with Bonferroni's multiple comparison test. ( H ) Quantification of IR-induced γH2AX foci in prostate cancer cell lines with different ID3 expression levels treated with 2 Gy, ca. 500 cells were counted at the indicated time points. n = 3 independent experiments; data are presented as mean ± SEM, One-Way ANOVA with Bonferroni's multiple comparison test. ( I , J ) NHEJ efficiency measured in U2OS-EJ5 cells and HR efficiency measured in U2OS-DR cells using the indicated siRNAs, respectively. n = 3 independent experiments; data are presented as mean ± SD, one-way ANOVA withTukey's multiple comparison test. ( K ) Western blot of the chromatin-bound fractions of RAD50, NBS1 and RECQL in U2OS cells (Representative Western blot, left panel) and a bar plot of the quantification of three independent Western blots (right panel, mean ± SD). ( L ) Clonogenic survival assay of U2OS cells transfected with the indicated siRNAs and treated with 2Gy of ionizing radiation. n = 3 independent experiments; data are presented as mean ± SEM, one-way ANOVA with Bonferroni's multiple comparison test. ( M ) Representative Micrographs and quantification of IR-induced γH2AX foci in U2OS cells transfected the indicated siRNAs, ca. 500 cells are counted at indicated time points. n = 3 independent experiments; data are presented as mean ± SEM, one-way ANOVA with Bonferroni's multiple comparison test. Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Clonogenic Cell Survival Assay, Transfection, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 loss impacts HR. ( A , B ) Representative micrographs and quantification of IR-induced γH2AX foci in Du145 and U2OS cells counted in G1 and S/G2 cells using the cell cycle marker CenpF. n = 3 independent experiments; data are presented as mean ± SD, one-way ANOVA withTukey's multiple comparison test. ( C ) Enrichment of ID3 at NHEJ-prone and HR-prone DSBs in AID-DIvA cells, measured by ChIP-qPCR. n = 3 independent experiments; data are presented as mean ± SEM,Student's t test. ( D ) Western blot of the chromatin-bound fractions of DNA-PKcs, XRCC4 and pRPA (S4/S8) (representative Western blot, left panel) and a bar plot of the quantification of three independent Western blots (right panel, mean ± SD), Student's t test was performed. ( E ) Western blot of the chromatin-bound fractions of CtIP and MRE11 (representative Western blot, left panel) and a bar plot of the quantification of three independent Western blots (right panel, mean ± SD), Student's t test was performed. ( F , G ) Representative micrographs and quantification of IR-induced RAD51 foci in Du145 and U2OS cells, respectively, ca. 500 cells were counted at indicated time points. n = 3 independent experiments; data are presented as mean ± SEM, Student's t test was performed. ( H ) Representative micrographs and quantification of IR-induced RAD51 foci in Du145 and U2OS cells, respectively, ca. 500 cells were counted at indicated time points. n = 3 independent experiments; data are presented as mean ± SEM, Student's t test was performed. Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Marker, Western Blot

Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 regulates expression of genes involved in DNA repair in response to IR. ( A ) Volcano plots showing differentially expressed genes in ID3-KO cells; untreated (0 Gy, left) and irradiated cells (5 Gy, right). Measured by RNA-seq ( n = 4). ( B ) Gene ontology enrichment analysis of the significantly downregulated genes in irradiated ID3-KO cells. ( C ) Barcode plot showing gene set enrichment analysis of DNA repair pathways of differentially expressed repair genes in irradiated ID3-KO cells. NES = normalized enrichment score. ( D ) Heat map showing differentially expressed DNA repair genes with a log 2 fold change < -0.5 for downregulated genes and > 0.5 for upregulated genes in irradiated WT, ID3-KO and ID3-rescue cells. n = 4, 4 and 2 for WT, KO and rescue cells, respectively. ( E ) RT-qPCR expression analysis of BRCA1, EXO1, RBBP8 (CtIP) and RAD51 genes in WT and ID3-KO with IR treatment (5 Gy) at indicated time points, n = 5, data presented as mean ± SEM, one-way ANOVA with Dunnett's multiple comparison test to compare all to the WT untreated. ( F ) Western blot showing expression of BRCA1, EXO1, RBBP8 (CtIP), and RAD51 proteins in WT and ID3-KO with IR treatment at indicated time points (representative Western blot, upper panel) and a bar plot of the quantification of three independent Western blots (lower panel, mean ± SD), Student's t test was performed. Statistical significance is presented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Expressing, Irradiation, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 regulates the chromatin accessibility and activity of E2F1 TF which mediate expression of HR genes in response to IR. ( A ) Scatter plot showing the correlation between differential chromatin accessibility at promoter regions and gene expression in irradiated cells. ( B , C ) Mean of the chromatin accessibility at promoter regions of genes involved in HR and BER. n = 3 for WT and KO, Student's t test. ( D ) Transcription factor binding motif analysis in all downregulated genes in ID3-KO after IR. ( E ) DiffTF analysis showing the differential activity of TFs in irradiated cells, E2F TFs are highlighted. Blue area represents TFs with reduced activity, orange represents TFs with increased activity. ( F ) Correlation between the activity of E2F TFs and the expression of their target genes. ( G ) Enrichment of E2F1 at the promoters of the indicated genes in WT and ID3-KO cells, measured by ChIP-qPCR. n = 2 independent experiments; data are presented as mean of six technical replicates from two independents experiments ± SEM, Student's t test. ( H , I ) RT-qPCR expression analysis of PRMT5 and TFDP1 genes in WT and ID3-KO with IR treatment at indicated time points, n = 5, data presented as mean ± SEM, one-way ANOVA with Dunnett's multiple comparison test to compare all to the WT untreated. ( J ) Western blot showing expression of PRMT5 and DP1 proteins in WT and ID3-KO with IR treatment at indicated time points (Representative Western blot, left panel) and a bar plot of the quantification of three independent Western blots (right panel, mean ± SD), Student's t test was performed. Statistical significance is presented as: * P < 0.05 , **P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Activity Assay, Expressing, Irradiation, Binding Assay, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition

doi: 10.1093/nar/gkab964

Figure Lengend Snippet: ID3 low expression is associated with impaired HR in cancer patients. ( A ) Box plot representation showing the single sample GSVA enrichment score of GO: regulation of double strand break via homologous repair in clusters of patient samples of TCGA prostate adenocarcinoma (PRAD). ( B ) Box plot representation showing the single sample GSVA enrichment score of GO: double strand break repair via break-induced replication in clusters of patient samples of TCGA PRAD. ( C ) Box plot representation showing ID3 expression as log 2 counts per million in clusters of patient samples of TCGA PRAD. ( D ) Box plot representation showing the single sample GSVA enrichment score of GO: regulation of double strand break via homologous repair in clusters of patient samples of TCGA testicular germ cell tumors (TGCT). ( E ) Box plot representation showing the single sample GSVA enrichment score of GO: double strand break repair via break-induced replication in clusters of patient samples of TCGA TCGT. ( F ) Box plot representation showing ID3 expression as log 2 counts per million in clusters of patient samples of TCGA TGCT. ( G ) Box plot representation showing the single sample GSVA enrichment score of GO: regulation of double strand break via homologous repair in clusters of patient samples of TCGA low grade glioma (LGG). ( H ) Box plot representation showing the single sample GSVA enrichment score of GO: double strand break repair via break-induced replication in clusters of patient samples of TCGA LGG. ( I ) Box plot representation showing ID3 expression as log 2 counts per million in clusters of patient samples of TCGA LGG patient samples. Group comparisons were performed using an unpaired Wilcoxon rank test. The dotted lines in all panels represent the average score across all groups. All group comparisons were performed using an unpaired Wilcoxon rank test. ( J ) Bubble plot representation showing the Pearson correlation coefficient between the expression of DNA repair genes ( BRCA1 , BRCA2 , RAD51 , EXO1 , RBBP8 , FANCM , PRMT5 , TFDP1 and E2F1 ) and ID3 in clusters of TCGA patient samples of PRAD (top), TGCT (middle) and LGG (bottom). Empty squares represent no significant correlation ( P > 0.05). The color of the circles represent the degree of correlation (red: positive; blue: negative) and the larger the size of the circle the stronger the significance.

Article Snippet: The ID3 full length, phospho-mutant ID3-S65E and -S65A, and empty-pLVX vector were cloned into XhoI- BamHI sites of pLVX-Puro to form ID3-pLVX (provided by Biocat).

Techniques: Expressing