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

Journal: Nucleic Acids Research

Article Title: CARM1/PRMT4 facilitates XPF–ERCC1 heterodimer assembly and maintains nucleotide excision repair activity

doi: 10.1093/nar/gkaf355

Figure Lengend Snippet: Antibody

Article Snippet: ORC2 , Santa Cruz Biotechnology, Dallas, TX, USA , sc-13238 , .

Techniques:

Journal: Nucleic Acids Research

Article Title: CARM1/PRMT4 facilitates XPF–ERCC1 heterodimer assembly and maintains nucleotide excision repair activity

doi: 10.1093/nar/gkaf355

Figure Lengend Snippet: CARM1 deficiency suppresses XPF–ERCC1 accumulation at DNA damage sites and delays CPD removal. ( A ) CARM1 KO inhibited CPD removal ability. The defect of CPD removal was rescued by exogenous CARM1-WT but not by inactivated CARM1-AAA. Data are shown as the mean ± SD of biological three independent experiments (multiple comparisons: two-way ANOVA, ** P < .01, **** P < .0001). ( B ) CARM1 KO and KD cells showed increased sensitivity to UV light. Biological three independent experiments were performed. Error bars represent SD * P < .05, ** P < .01, and *** P < .001 (Student’s t -test). ( C ) Accumulation of XPC at local UV-exposed sites was not different between siCt and siCARM1, but XPF and ERCC1 accumulations were inhibited by siCARM1. Representative images of immunostaining for XPC and XPF and phosphor HBO1 and ERCC1 in local UV-irradiated HeLa cells. HeLa cells were irradiated with 50 J m −2 UV through an 8-μm pore membrane and fixed after 30 min of incubation. The mean intensity of the irradiated area was divided by the intensity of the non-irradiated area of the same nucleus and expressed as a relative intensity increase from the baseline (%). Scale bars, 5 μm. Data are shown as the mean (unpaired t -test). ( D ) CARM1 interacts with XPF in HEK293 cells. ( E ) XPF interacts with CARM1 in DOX-induced XPF stable expression cells and 293 cells. ( F ) XPF WT-Myc and GST-CARM1 interact in a GST pull-down assay. When XPF WT-Myc overexpressed in HEK293 cells was pulled down using GST-CARM1 expressed and purified in E. coli , XPF WT-Myc specifically co-precipitated. *An unknown E. coli protein that is co-purified when GST-CARM1 is purified from E. coli . ( G ) CARM1 KD cells showed decreased total protein content of XPF and decreased chromatin localization. XPF amounts were corrected for ORC2. The amount of XPF in each fraction is expressed as relative amount (%) to the siCt soluble fraction without UV. Biological five independent experiments were performed. Error bars represent SD. * P < .05 and *** P < .001 (Student’s t -test).

Article Snippet: ORC2 , Santa Cruz Biotechnology, Dallas, TX, USA , sc-13238 , .

Techniques: Immunostaining, Irradiation, Membrane, Incubation, Expressing, Pull Down Assay, Purification

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Western Blot, Staining, Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Staining, Knock-Out, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Staining

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Mutagenesis, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques: Two Tailed Test, Staining, Quantitation Assay, Imaging

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.

Article Snippet: Mouse ORC2 antibody was raised against His tagged full length of ORC2 recombinant protein in Rabbit (Pacific Immunology).

Techniques:

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Western Blot, Staining, Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Staining, Knock-Out, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Staining

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Mutagenesis, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques: Two Tailed Test, Staining, Quantitation Assay, Imaging

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.

Article Snippet: Mice with LoxP sites inserted flanking exons 6 and 7 of mouse Orc2 were purchased from Cyagen Biosciences Inc ( ).

Techniques:

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of introduced loxP sites in Orc 2 locus. ( B ) Representative picture of genotyping of offspring coming from Orc2 f/+ crossed with Orc2 f/+ . ( C ) The ratio of observed to expected animals coming from Orc2 f/+ crossed with Orc2 f/+ . ( D ) Schematic of the ORC2 protein and the DeltaORC2 protein produced after deletion of exons 6 and 7. A110 is mutated to V110 and then the protein goes out of frame. ( E ) Validation of Orc2 deletion 3 d after Adeno cre transduction. ( F ) Western blot of ORC2 protein 5 d after Adeno cre transduction. 10 or indicated μl of lysate loaded/lane as written on the top. ( G ) MTT assay of WT and Orc2 f/f MEFs without and with Adeno cre transduction. ( H ) Western blot of ORC2 protein 5 and 15 d after Adeno Cre transduction. Figure 1—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 1—source data 2. Original image for , panel B. Figure 1—source data 3. PDF file containing original DNA gel picture corresponding to , panel E, indicating the relevant bands and increasing Adeno-Cre. Figure 1—source data 4. Original image for , panel E. Figure 1—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel F, indicating the relevant bands and addition of Adeno-Cre. Figure 1—source data 6. Original image for , panel F. Figure 1—source data 7. PDF file containing original Western blot membrane picture corresponding to , panel H, indicating the relevant bands and ORC2 protein expression. Figure 1—source data 8. Original image for , panel H.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Produced, Biomarker Discovery, Transduction, Western Blot, MTT Assay, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: Embryonic lethality of Orc2 KO . The Orc2Δ allele was created by expressing Cre recombinase from a Sox2 promoter in the Orc2 f/f embryos.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Scheme of Alb +/- - Orc2 f/f ROSA26 stop-EYFP crossed with Alb +/- -Orc2 f/f ROSA26 stop-EYFP (All mice are with ROSA26 stop-EYFP and so we do not include this in the genotypes below). ( B ) The ratio of observed to expected animals coming from A. ( C ) Western blot of hepatocytes from Orc2 f/f and Alb +/- - Orc2 f/f animals. Tubulin was used as loading control. ( D ) Quantification of the Western blots of hepatocyte lysates from Orc2 f/f (without Alb-cre ) mice and the same genotype but with Alb-Cre to show the levels of other key replication initiation proteins in the ORC2 KO hepatocytes. ( E ) Average body weight of Orc2 f/f and Alb-Orc2 f/f animals. ( F ) Average liver weight of Orc2 f/f and Alb-Orc2 f/f animals. ( G ) Average liver-to-body weight ratio of Orc2 f/f and Alb-Orc2 f/f animals. ( H ) Representative H&E staining of liver tissue from Orc2 f/f (WT) and Alb-Orc2 f/f (KO) animals. Both panels at same scale. ( I ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f animals. ( J ) Quantification of hepatocyte nuclear size in Orc2 f/f and Alb-Orc2 f/f female mice. ( K ) Quantification of hepatocytes nuclear size in Orc2 f/f and Alb-Orc2 f/f male mice. *p<0.05, **p<0.01, two-tailed Student’s t-test. Figure 2—source data 1. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent ORC2 protein. Figure 2—source data 2. Original image for , panel C. Figure 2—source data 3. Original Western blot membrane picture corresponding to , panel C. Molecular weight markers are labeled on the left. The bands next to the arrow represent Tubulin protein. Figure 2—source data 4. Original image for , panel C.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Western Blot, Control, Staining, Two Tailed Test, Membrane, Molecular Weight, Labeling

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Experimental design. ( B–D ) Quantification of nuclei ploidy on 10,000 nuclei from the livers of Orc2 f/f ROSA26 stop-EYFP and Alb-Orc2 f/f ROSA26 stop-EYFP animals. ( E–G ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Genotyping and western blotting of hepatocytes. ( C ) Representative picture of EdU, EYFP and DAPI staining on the Orc2 WT ( Orc2 f/f ) and KO ( Orc2 f/f Alb-Cre ) primary hepatocytes. ( D ) The percentage of EdU positive nuclei from Orc2 WT or Orc2 KO primary hepatocytes. *p < 0.05, two-tailed Student’s t test. Figure 4—source data 1. PDF file containing original DNA gel picture corresponding to , panel B, indicating the relevant bands and individual animals. Figure 4—source data 2. Original image for , panel B.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Western Blot, Staining, Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Schematic of the experiment. ( B ) Body weight of the Orc2 f/f ROSA26 stop-EYFP mice without (-/-) or with Alb-Cre (+/-) before partial hepatectomy. ( C ) Liver weight of the mice in B after liver regeneration. ( D ) Regenerated liver to pre-hepatectomy body weight ratio of the mice in B. ( E ) H&E stain of Orc2 f/f ROSA26 stop-EYFP livers with intact Orc2 ( Alb-cre -/- , N=3) or Orc2 knockout ( Alb-cre +/- , N=7). Scale bar: 25 μm. ( F ) Quantitation of nuclear counts per field (76,000 μm 2 ). Six images were taken for each liver. 0 hr (pre-resection). 42 hr (post-regeneration). ( G ) EdU incorporation of indicated livers. EYFP marks cells where Cre has been expressed. Orc2 ( Alb-cre -/- , N=5) or Orc2 knockout ( Alb-cre +/- , N=5). Scale bar: 25 μm. ( H ) Percent EdU+ nuclei counted in 1882 and 825 nuclei in the Cre- and Cre+ livers, respectively. ( I ) Nuclear size of indicated livers. 0 hr (pre-resection). 42 hr (post-regeneration). Mean and S.D from about 40–70 nuclei, *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t test is used.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Staining, Knock-Out, Quantitation Assay, Two Tailed Test

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–B ) Breeding schemes to obtain conditional double flox animals. ( C ) The ratio of observed to expected animals coming from B. Orc1 =all animals with Orc1 f/f ROSA26 stop-EYFP , Orc2 =all animals with Orc2 f/f ROSA26 stop-EYFP , Orc1 Orc2 =all animals with Orc1 f/f Orc2 f/f ROSA26 stop-EYFP genotype. This was before the introduction of Alb-Cre . ( D ) Immunoblot of hepatocytes from WT ( Orc1 f/f Orc2 f/f ) and DKO ( Orc1 f/f Orc2 f/f Alb-cre +/- ) mice to show that ORC1 and ORC2 are depleted in the DKO cells. ( E ) Quantitation of immunoblots to show that levels of other key initiation protein subunits are not decreased in the DKO mice hepatocytes. ( F ) Average body, liver weight, and their ratio for WT and DKO animals. ( G ) Representative H&E staining of liver tissue from male WT and DKO animals. ( H ) Quantification of hepatocyte nuclear size in the WT and DKO animals. ( I ) Quantification of nuclei ploidy for EYFP low (includes negative) and high (positive) primary liver cells from DKO mice. Figure 6—source data 1. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC1 protein expression, and individual animals. Figure 6—source data 2. Original image for panel D, ORC1 protein expression, and individual animals. Figure 6—source data 3. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, ORC2 protein expression, and individual animals. Figure 6—source data 4. Original image for panel D, ORC2 protein expression, and individual animals. Figure 6—source data 5. PDF file containing original Western blot membrane picture corresponding to , panel D, indicating the relevant bands, HSP90 protein expression, and individual animals. Figure 6—source data 6. Original image for panel D, HSP90 protein expression, and individual animals.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Western Blot, Quantitation Assay, Staining, Membrane, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Representative picture of livers of female Orc1 Orc2 WT and liver-specific double KO (dKO). ( B ) Representative H&E staining of liver tissue from Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (WT) and Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females. ( C ) Kaplan-Meier plot for Alb-Orc1 f/f Orc2 f/f ROSA26 stop-EYFP (dKO) females postnatally.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Staining

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A ) Ratio of liver weight to body weight for mice at 6 wk of age. The Orc1 mutant, Orc2 mutant or double mutant mice are in red. ( B ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of one ORC subunit are floxed (underlined): Orc1 f/f or Orc2 f/f . ( C ) Enlarged nuclei seen in 6 wk mouse livers in mice expressing Alb-Cre where both alleles of two ORC subunits are floxed (underlined): Orc1 f/f Orc2 f/f .

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Mutagenesis, Expressing

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: ( A–C ) Body weight pre-resection, liver weight post-regeneration, and regenerated liver to body weight ratio in mice with indicated genotypes. Black bars: 4 wild-type (WT) males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice without Alb-Cre ). White bars: 6 dKO males ( Orc1 f/f Orc2 f/f ROSA26 stop-EYFP mice with Alb-Cre +/- ). No significant difference between the two groups using two-tailed Student t-test. ( D ) H&E stain of WT (N=4) or DKO mice (N=6). Scale bar: 50 μm. ( E ) Quantitation of hepatocyte nuclear size post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. About 120–200 nuclei are counted. ( F ) Quantitation of hepatocyte nuclear density post regeneration. WT: black bars. DKO: white bars. 0 hr (pre-resection). 42 hr (post-regeneration). Five-six images were taken for each liver. ( G ) Micrographs of EdU, DAPI and EYFP imaging of livers with indicated genotypes post regeneration. WT (N=6) and DKO mice (N=7). Scale bar: 20 μm. WT in the top row, DKO in the bottom row. ( H ) Quantitation of EdU positive nuclei post regeneration. WT: black bar. DKO: white bar. Five-six images were taken for each liver. *p<0.05, ****p<0.0001, unpaired two-tailed Student’s t-test were used.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: Two Tailed Test, Staining, Quantitation Assay, Imaging

Journal: eLife

Article Title: DNA replication in primary hepatocytes without the six-subunit ORC

doi: 10.7554/eLife.102915

Figure Lengend Snippet: Estimate of number of hepatocyte nuclei in adult mice of indicate genotypes and thus, number of hepatocyte nuclear divisions required after E9.5 mouse embryos.

Article Snippet: Orc2 f/f mice were generated by Cyagen Biosciences Inc Exons 6–7 (amino acids L111-V150) was selected as conditional knockout region (cKO).

Techniques: