Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Subcellular fractionation and western blotting of WT, ORC2Δ , and ORC2 rescue cells. S2, cytoplasmic fraction; S3, nuclear soluble fraction; P3, nuclear insoluble fraction. Asterisk indicates non-specific signal. (B) HP1 foci are unchanged in ORC2Δ cells. Scale bar: 10 μm. (C) H3K9me3 ChIP-seq peaks altered in KO cells and rescued by re-introduction of ORC2. (D) Example H3K9me3 peaks that are dependent on ORC2 and down in the KO (blue) or up in the KO (purple) ( n = 2). (E) ChIP qPCR shows that ORC2 KO does not change H3K9me3 signal on telomeres or centromeres ( n = 3 technical replicates). Data are represented as mean ± SD. (F) Number of bases covered by ORC2-dependent H3K9me3 and K27me3. Red, upregulated in KO and rescued; blue, downregulated in KO and rescued. See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: Fractionation, Western Blot, ChIP-sequencing, ChIP-qPCR

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) ATAC-seq peak signals compared in WT and ORC2Δ cells. The significantly up (red) and down (blue) peaks in ORC2Δ are indicated (adjusted p < 0.05, fold change more than 2-fold). (B and C) Heatmap of ATAC signal in the three matched cell lines centered on peaks (B) upregulated or (C) downregulated in the KO cells ( n = 2). (D) Number of ATAC peaks altered in the ORC2Δ . Red indicates the sites where the changes were reversed with re-introduction of ORC2. (E) Enrichment of ORC regulated ATAC peaks in enhancers. (F and G) (F) H3K9me3 and (G) H3K27me3 signal distribution on ATAC-up or ATAC-down peaks in three cell lines. *** p < 0.001 by Student’s t test compared to ORC2Δ . (H and I) Heatmap of ATAC signal centered on (H) centromeres and (I) telomeres in three cell lines. (J) qPCR of ATAC-positive DNA at centromeres of three chromosomes ( n = 3 technical replicates). Data are represented as mean ± SD. See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques:

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) RNA-seq signal of different genes plotted to show mean expression and fold change in ORC2Δ relative to WT ( n = 3). Genes significantly upregulated (red) and downregulated (blue) in the ORC2Δ . (B) Number of genes whose expression is altered in the ORC2Δ . Red indicates genes whose expression change is reversed upon re-introduction of ORC2. (C and D) Heatmap of expression levels of genes that are (C) upregulated or (D) downregulated in the ORC2Δ and rescued ( n = 3). (E) Expression changes of genes in ORC2Δ cells correlated with change of ATAC accessibility in the TSS region. (F and G) (F) CHD7 and (G) NFIA RNA downregulated in an ORC2-dependent manner ( n = 3). Global comparison was by one-way ANOVA test across three cell lines, and pairwise comparison with ORC2Δ was done with Student’s t test (*** p < 0.001, * p < 0.05). (H) Western blot shows CHD7 and NFIA protein decrease on chromatin-bound frac in ORC2Δ and rescue by ORC2. (I and J) H3K27me3 ChIP-seq signal over the (I) CHD7 and (J) NFIA gene in the three cell lines ( n = 2). See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: RNA Sequencing, Expressing, Comparison, Western Blot, ChIP-sequencing

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Schematic of experiment. (B and C) Chromatin-accessibility (ATAC-seq) and gene-expression (RNA-seq) changes in ORC1Δ . Signals significantly upregulated (red) or downregulated (blue) in the ORC1Δ cells. (D and E) Same as (B) and (C) except for ORC5Δ cells relative to WT. (F and G) Venn diagrams showing overlap of ATAC changes in the three KO cells. (H) Example ORC2-dependent ATAC changes (chr19: 608305–614483; chr2: 28407886–2841 1076; chr13: 21031256–21037283; chr17: 72,698,613–72,700,987). (I and J) Venn diagram showing overlap of gene-expression changes in the three KO cells. See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Schematic of ChEC-seq experiments to identify binding sites of ORC subunits. (B) Number of peaks bound by each of the three ORC subunits and their overlap. (C and D) Heatmap of binding of the three subunits centered on sites bound by (C) ORC2 or (D) TSSs ( n = 2). (E) Enrichment of binding sites of ORC subunits on enhancers and promoters. (F) ORC2-binding sites are enriched at sites that become more accessible (ATAC-up) when ORC2 is lost. A lesser enrichment is seen at ATAC-down sites. Z and p value are derived from permutation test ( n = 1,000), *** p < 0.001. (G) BART analysis of transcription factors whose known-binding sites are enriched near ORC2-binding sites. See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Example compartment changes on Chr 16 from A (red) to B in the ORC2Δ cells and is rescued by ORC2 restoration. The H3K9me3 and H3K27me3 tracks ( n = 2) are shown. (B) Changes in PC1 when the KO is compared to the WT ( x axis) or the rescue ( y axis) are correlated with each other at many more sites than show sign (compartment) changes. Colors of the dots represent compartment status in the ORC2Δ (red, A compar; cyan, B). (C) Number of loops that increase or decrease in the KO relative to WT, and the portion that is rescued by ORC2 restoration. (D) Heatmap of loop strength of the loops (up or down) that are rescued in (C). (E) Loops that become stronger in the KO are longer than the loops that become weaker. (F) Enrichment of ORC2-binding sites across the region centered on loop-up or loop-down sites. (G) Enrichment of loop-up anchors across the region centered on loop-down anchors. See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: Binding Assay

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Venn diagram of CTCF-binding sites in the three cell lines. Red, CTCF-binding sites that are increased in the KO and decreased in the rescue; blue, CTCF-binding sites present in the WT that disappeared in the KO and re-appeared when ORC2 is restored. (B) CTCF motif enrichment ratio in indicated classes of CTCF-binding sites. (C) Enrichment of ORC2-binding sites across the region centered on CTCF-binding sites of the three classes by permutation test ( n = 1,000). (D) Enrichment of ATAC-up or -down sites at CTCF-binding sites of the three classes by permutation test ( n = 1,000). (E) Top three tracks: Loops in the NFIA locus in the three cell lines. The loop anchor sites a–f are indicated at the top and marked by the colored bars running through all tracks. ORC2- and CTCF-binding sites are shown in the middle tracks. Bottom three tracks: ORC2-dependent prevention of H3K27me3 mark at the locus. See also .

Article Snippet: Antibodies used include α-ORC1 (cell signaling technology, no. 4731), α-ORC2 (Santa Cruz Biotechnology, no. sc-32734), α-ORC3 (Santa Cruz Biotechnology, no. sc-23888), α-ORC4, α-ORC5, α-ORC6 (home-made ), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (a generous gift from Supriya G. Prasanth, University of Illinois ), α-HP1α (cell signaling technology, no. 2616), α-H3K9ME1 (cell signaling technology, no. 14186), α-H3K9ME2 (cell signaling technology, no. 4658), α-H3K9ME3 (cell signaling technology, no. 13969), α-HBO1 (cell signaling technology, no. 58418), α-H3K27ME3 (cell signaling technology, no. 9733), α-Histone H3 (cell signaling technology, no. 4499), α-NFIA (Abcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: Binding Assay

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Subcellular fractionation and western blotting of WT, ORC2Δ , and ORC2 rescue cells. S2, cytoplasmic fraction; S3, nuclear soluble fraction; P3, nuclear insoluble fraction. Asterisk indicates non-specific signal. (B) HP1 foci are unchanged in ORC2Δ cells. Scale bar: 10 μm. (C) H3K9me3 ChIP-seq peaks altered in KO cells and rescued by re-introduction of ORC2. (D) Example H3K9me3 peaks that are dependent on ORC2 and down in the KO (blue) or up in the KO (purple) ( n = 2). (E) ChIP qPCR shows that ORC2 KO does not change H3K9me3 signal on telomeres or centromeres ( n = 3 technical replicates). Data are represented as mean ± SD. (F) Number of bases covered by ORC2-dependent H3K9me3 and K27me3. Red, upregulated in KO and rescued; blue, downregulated in KO and rescued. See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques: Fractionation, Western Blot, ChIP-sequencing, ChIP-qPCR

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) ATAC-seq peak signals compared in WT and ORC2Δ cells. The significantly up (red) and down (blue) peaks in ORC2Δ are indicated (adjusted p < 0.05, fold change more than 2-fold). (B and C) Heatmap of ATAC signal in the three matched cell lines centered on peaks (B) upregulated or (C) downregulated in the KO cells ( n = 2). (D) Number of ATAC peaks altered in the ORC2Δ . Red indicates the sites where the changes were reversed with re-introduction of ORC2. (E) Enrichment of ORC regulated ATAC peaks in enhancers. (F and G) (F) H3K9me3 and (G) H3K27me3 signal distribution on ATAC-up or ATAC-down peaks in three cell lines. *** p < 0.001 by Student’s t test compared to ORC2Δ . (H and I) Heatmap of ATAC signal centered on (H) centromeres and (I) telomeres in three cell lines. (J) qPCR of ATAC-positive DNA at centromeres of three chromosomes ( n = 3 technical replicates). Data are represented as mean ± SD. See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques:

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) RNA-seq signal of different genes plotted to show mean expression and fold change in ORC2Δ relative to WT ( n = 3). Genes significantly upregulated (red) and downregulated (blue) in the ORC2Δ . (B) Number of genes whose expression is altered in the ORC2Δ . Red indicates genes whose expression change is reversed upon re-introduction of ORC2. (C and D) Heatmap of expression levels of genes that are (C) upregulated or (D) downregulated in the ORC2Δ and rescued ( n = 3). (E) Expression changes of genes in ORC2Δ cells correlated with change of ATAC accessibility in the TSS region. (F and G) (F) CHD7 and (G) NFIA RNA downregulated in an ORC2-dependent manner ( n = 3). Global comparison was by one-way ANOVA test across three cell lines, and pairwise comparison with ORC2Δ was done with Student’s t test (*** p < 0.001, * p < 0.05). (H) Western blot shows CHD7 and NFIA protein decrease on chromatin-bound frac in ORC2Δ and rescue by ORC2. (I and J) H3K27me3 ChIP-seq signal over the (I) CHD7 and (J) NFIA gene in the three cell lines ( n = 2). See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques: RNA Sequencing, Expressing, Comparison, Western Blot, ChIP-sequencing

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Schematic of experiment. (B and C) Chromatin-accessibility (ATAC-seq) and gene-expression (RNA-seq) changes in ORC1Δ . Signals significantly upregulated (red) or downregulated (blue) in the ORC1Δ cells. (D and E) Same as (B) and (C) except for ORC5Δ cells relative to WT. (F and G) Venn diagrams showing overlap of ATAC changes in the three KO cells. (H) Example ORC2-dependent ATAC changes (chr19: 608305–614483; chr2: 28407886–2841 1076; chr13: 21031256–21037283; chr17: 72,698,613–72,700,987). (I and J) Venn diagram showing overlap of gene-expression changes in the three KO cells. See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques: Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Schematic of ChEC-seq experiments to identify binding sites of ORC subunits. (B) Number of peaks bound by each of the three ORC subunits and their overlap. (C and D) Heatmap of binding of the three subunits centered on sites bound by (C) ORC2 or (D) TSSs ( n = 2). (E) Enrichment of binding sites of ORC subunits on enhancers and promoters. (F) ORC2-binding sites are enriched at sites that become more accessible (ATAC-up) when ORC2 is lost. A lesser enrichment is seen at ATAC-down sites. Z and p value are derived from permutation test ( n = 1,000), *** p < 0.001. (G) BART analysis of transcription factors whose known-binding sites are enriched near ORC2-binding sites. See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques: Binding Assay, Derivative Assay

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Example compartment changes on Chr 16 from A (red) to B in the ORC2Δ cells and is rescued by ORC2 restoration. The H3K9me3 and H3K27me3 tracks ( n = 2) are shown. (B) Changes in PC1 when the KO is compared to the WT ( x axis) or the rescue ( y axis) are correlated with each other at many more sites than show sign (compartment) changes. Colors of the dots represent compartment status in the ORC2Δ (red, A compar; cyan, B). (C) Number of loops that increase or decrease in the KO relative to WT, and the portion that is rescued by ORC2 restoration. (D) Heatmap of loop strength of the loops (up or down) that are rescued in (C). (E) Loops that become stronger in the KO are longer than the loops that become weaker. (F) Enrichment of ORC2-binding sites across the region centered on loop-up or loop-down sites. (G) Enrichment of loop-up anchors across the region centered on loop-down anchors. See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques: Binding Assay

Journal: Cell reports

Article Title: Regulation of epigenetics and chromosome structure by human ORC2

doi: 10.1016/j.celrep.2025.115816

Figure Lengend Snippet: (A) Venn diagram of CTCF-binding sites in the three cell lines. Red, CTCF-binding sites that are increased in the KO and decreased in the rescue; blue, CTCF-binding sites present in the WT that disappeared in the KO and re-appeared when ORC2 is restored. (B) CTCF motif enrichment ratio in indicated classes of CTCF-binding sites. (C) Enrichment of ORC2-binding sites across the region centered on CTCF-binding sites of the three classes by permutation test ( n = 1,000). (D) Enrichment of ATAC-up or -down sites at CTCF-binding sites of the three classes by permutation test ( n = 1,000). (E) Top three tracks: Loops in the NFIA locus in the three cell lines. The loop anchor sites a–f are indicated at the top and marked by the colored bars running through all tracks. ORC2- and CTCF-binding sites are shown in the middle tracks. Bottom three tracks: ORC2-dependent prevention of H3K27me3 mark at the locus. See also .

Article Snippet: ORC2 (3G6) Rat mAb , Santa Cruz Biotechnology , Cat #sc-32734; RRID: AB_2157726.

Techniques: Binding Assay

Journal: Advanced Science

Article Title: Topoisomerase I Inhibition in ETV4‐overexpressed Non‐Small Cell Lung Cancer Promotes Replication and Transcription Mediated R‐Loop Accumulation and DNA Damage

doi: 10.1002/advs.202409307

Figure Lengend Snippet: ETV4 transcriptionally controls key genes involved in DNA replication of NSCLC cells. A) Functional analysis showing the top KEGG pathways that are significantly associated with down‐regulated genes in H1299, H1703, and H358T si‐ETV4 groups normalized to negative control (NC) siRNA groups by Human Microarray ( GSE137445 ). B) Clustering analysis showing the gene signature is enriched in the DNA replication pathway. C) IGV tracks showing the enrichment of ETV4 at the promoter region of MCM2, ‐4, ‐5, ‐10, and ORC1 from ChIP‐seq signals of A549‐shETV4 cells transfected with Flag‐ETV4 plasmids. D) ChIP‐PCR analysis for endogenous or exogenous ETV4 binding to the promoter region of MCM2, ‐4, ‐5, ‐10, and ORC1 genes in H1299 cells using anti‐ETV4 antibody or H358‐ETV4 cells using anti‐Flag antibody. E) Schematic depicting the core motif of ETV4 binding with its target genes, the wild‐type luciferase reporter constructs, and the mutations in the putative ETV4‐binding sites (mut‐1 or mut‐2 type) of MCMs/ORC1 promoter region. F–J) The wild‐type luciferase reporter plasmids of MCMs (ORC1) were co‐transfected along with ETV4, ETV4‐DBD deletion plasmid, or the empty vector into HEK293T cells. The constructed luc‐MCMs (ORC1)‐mut‐1 or ‐mut‐2 type plasmids were co‐transfected along with ETV4 plasmid or the empty vector into HEK293T cells. Relative luciferase activity was normalized against Renilla luciferase activity, respectively (mean ± SD; n = 4; ordinary one‐way ANOVA with Tukey's multiple comparisons test). **** p < 0.0001; ### p < 0.001, #### p < 0.0001. K) RT‐qPCR analysis of MCM2, ‐4, ‐5, ‐10, and ORC1 mRNA expression in H1299 cells transfected with ETV4 or NC siRNA, Transcript levels were normalized to ACTB gene expression (mean ± SD, n = 3; two‐tailed unpaired t ‐test). ** p < 0.01; *** p < 0.001; **** p < 0.0001. L) Immunoblots showing MCM2, ‐4, ‐5, ‐10, and ORC1 protein levels in NC and ETV4‐knockdown H1299 cells. M,N) Immunoblots showing the Chromatin‐bound proteins (Chrom.) and unbound proteins (Sol.) levels of MCM2, ‐4, ‐5, ‐10, and ORC1 protein in control and sh‐ETV4 A549 cells, or control and ETV4‐overexpression H358 cells.

Article Snippet: Blots were blocked with 5% nonfat milk in Tris‐Buffered Saline and Tween 20 (TBST), and incubated with antibodies specific for ETV4 (10684‐1‐AP, Proteintech), MCM2 (3619, CST), MCM3 (15597‐1‐AP, Proteintech), MCM4 (13043‐1‐AP, Proteintech), MCM5 (11703‐1‐AP, Proteintech), MCM6 (13347‐2‐AP, Proteintech), MCM7 (11225‐1‐AP, Proteintech), MCM10 (12251‐1‐AP, Proteintech), ORC1 (NBP100‐121, Novus), ORC2 (12739‐1‐AP, Proteintech), ORC3 (sc‐374231, Santa Cruz), ORC4 (13026‐1‐AP, Proteintech), ORC5 (11542‐1‐AP, Proteintech), ORC6 (17784‐1‐AP, Proteintech), Histone‐H3 (17168‐1‐AP, Proteintech), PCNA (13110, CST), phospho‐MCM2 S40 (ab133243, Abcam), CDC45 (15678‐1‐AP, Proteintech), SUPT16H (28598‐1‐AP, Proteintech), SSRP1 (15696‐1‐AP, Proteintech), γ‐H2AX (ET‐1602, HUABio), Flag (14793, CST), His (66005‐1‐Ig, Proteintech), HA (sc‐7392, Santa Cruz), GST (66001‐2‐lg, Proteintech), GAPDH (60004‐1‐Ig, Proteintech) and β‐actin (66009‐1‐Ig, Proteintech) was used as a loading control for Western blot.

Techniques: Functional Assay, Negative Control, Microarray, ChIP-sequencing, Transfection, Binding Assay, Luciferase, Construct, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Expressing, Gene Expression, Two Tailed Test, Western Blot, Knockdown, Control, Over Expression