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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

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: ( 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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

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–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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

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: The antibodies used in this study are listed: ORC1 (Santa Cruz; sc-28741); ORC3 (Santa Cruz; sc-374231); ORC5 (Boster Biological technology; A03676-1); ORC6 (Santa Cruz; sc-390490); CDC6 (Santa Cruz; sc-9964); MCM2 (Abcam; ab4461); MCM3 (Santa Cruz; sc-9850).

Techniques:

Journal: bioRxiv

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

doi: 10.1101/2024.12.18.629220

Figure Lengend Snippet: (A) Schematic of experiment. (B-C) Chromatin accessibility (ATAC-seq) and gene expression (RNA-seq) changes in ORC1KO. Signals significantly up-regulated (red) or down-regulated (blue) in the ORC1 KO cells. (D-E) Same as (B-C) except for ORC5KO cells relative to WT. (F-G) Venn diagram showing overlap of ATAC changes in the three KO cells. (H) Example ORC2-dependent ATAC changes. (I-J) Venn diagram showing overlap of gene expression changes in the three KO cells.

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 (PMID15944161), α-HSP90 (Santa Cruz Biotechnology, no. sc-13119), α-MCM7 (Santa Cruz Biotechnology, no. sc-9966), α-ORCA (PMID 20932478, 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 (Apcepta, no. AP20998c), α-CHD7 (cell signaling technology, no. 6505).

Techniques: Gene Expression, RNA Sequencing