Fig 2: Mcm7 knockdown in zebrafish. (A) RT-PCR of control injected (CTRL MO) and Mcm7 MO injected embryos at 24 hpf. In embryos with Mcm7 MO the original band at 340 bp partially disappeared. Instead a second band around 1500 bp could be detected, which contained intronic sequences. n = 3 independent experiments. (B) Live images of zebrafish at 48 hpf. Scale bars: 500 µm. (C) Mcm7 depleted embryos develop smaller anterior structures, which is partially rescued by co-injection of RNA encoding human MCM7. Five independent experiments. **P < 0.01, ****P < 0.0001. One-way ANOVA with Holm–Sidak's multiple comparison test. (D) Loss of Mcm2 impairs eye development. Five independent experiments. ***P < 0.001, ****P < 0.0001. One-way ANOVA with Holm–Sidak's multiple comparison test. (E) Zebrafish lacking Mcm7 develop pericardial edema. 5 independent experiments. *P < 0.05, ***P < 0.0001. One-way ANOVA with Holm–Sidak's multiple comparison test. (C–E) Numbers of embryos analysed are given below the bars in E.

Fig 3: Morphological analysis of zebrafish lacking Mcm2. (A) Autoradiogram to show the binding efficiency of the Mcm2 MO. In vitro translation of Mcm2 was done in the presence of CTRL (CTRL MO) and translation blocking MO (Mcm2 MO) using a cell-free reticulocyte lysate and 35S-labeled methionine. As template, pCS2+ containing parts of the 5'-UTR fused to the ORF of Mcm2 was used. n = 3 MO binding tests (in triplicate). (B) RT-PCR of non-injected (NI), control injected (splCTRL) and splice blocking injected (splMO) embryos at 24 hpf. In embryos with Mcm2 splMO the original band at 322 bp partially disappeared. Instead a second band at 1685 bp could be detected, which contained parts of intron 2. n = 2 independent experiments. (C) Live images of zebrafish at 48 hpf. Scale bars: 500 µm. (D) Mcm2 depleted embryos develop smaller anterior structures. Numbers of embryos analysed are given below the bars. 3–10 independent experiments. **** indicates a P value =0.0001, *** means P = 0.001. One-way ANOVA with Sidak's multiple comparison test. (E) Loss of Mcm2 impairs eye development. Numbers of embryos analysed are given below the bars. 3–6 independent experiments. **** indicates a P value =0.0001, ** means P = 0.01. One-way ANOVA with Sidak's multiple comparison test. (F) Zebrafish lacking Mcm2 show a tendency to develop pericardial edema. Numbers of embryos analysed are given below the bars. 3–11 independent experiments. * indicates a P value <0.05. One-way ANOVA with Sidak's multiple comparison test.

Fig 4: Copy-numbers of amplified gene existing in DMs are decreased by cohesin reduction. (A) COLO 320-DM cells were stably transduced with control GFP-shRNA or two different Rad21-shRNAs (R#35 and R#98). Changes in copy-numbers of the c-Myc were quantified using a TaqMan quantitative PCR-based CNV assay. The average of three viral transduction experiments ± SD is presented. *P < 0.01. (B) Metaphase FISH analysis with c-Myc (labeled with FITC; green) and chromosome 2 centromere (labeled with Cy3 for the control; red) revealed a reduction of c-Myc located within DMs in RAD21-KD COLO 320-DM cells. (C) Total numbers of DMs per cell were blindly counted in control GFP-KD and RAD21-KD COLO 320-DM cells. (Mean ± SD, n = 50). *P < 0.01. (D) A ChIP was performed with antibodies to RAD21 and SMC1 in control GFP-KD (blue bar) or RAD21-KD COLO 320-DM (red bar). (Mean ± SEM, n = 3 biological replicates). 3'HS1 of the human ß-globin locus (amplicon 4) and Necdin (amplicon 5) served as controls. (E) 3C assay of c-Myc in COLO 320-DM cells after RAD21-KD. DpnII restriction sites in c-Myc appear as gray shaded bars. Black shading indicates the anchor fragment of the 5' CTCF binding sites of c-Myc. (Mean ± SEM, n = 3 biological replicates). (F) The expression of c-Myc was measured by qRT-PCR on day 60 after RAD21-KD in COLO 320-DM cells. Each value was normalized to that of 18S ribosomal RNA relative to the control GFP-shRNA expressing cells. The average of triplicate independent viral transduction experiments ± SD is presented. *P < 0.01. (G) A ChIP assay using previously published replication origin sequences near the c-Myc locus (70) was performed with antibodies against Pol a, CDC45 and MCM7 in control GFP-KD or RAD21-KD COLO 320-DM cells on day 60 after lentiviral transduction. (Mean ± SEM, n = 3 biological replicates).

Fig 5: Down-regulation of cohesin impairs localization of pre-replication complex at replication origins within the amplified APIP/PDHX/CD44 locus. (A) A ChIP assay was performed with SNU16 (blue bar) and HCT116 (red bar) cells using antibodies against RAD21, DNA polymerase a (Pol a), MCM7 and CDC45. The location of previously published replication origin sequences (70) near the APIP/PDHX/CD44 locus used for qPCR are shown with names below. (Mean ± SEM, n = 3 biological replicates). (B) A ChIP assay was performed with control GFP-KD (blue bar) or RAD21-KD SNU16 cells (R#35; yellow bar) on day 60 after lentivial transduction using antibodies against RAD21, Pol a, MCM7 and CDC45. (Mean ± SEM, n = 3 biological replicates). (C) Immunofluorescence staining of DNA (DAPI, blue), MCM7 (green) and RAD21 (red) in control GFP- KD (left panel) or RAD21-KD SNU16 cells (right panel). (D) Percentage of MCM7 and RAD21 co-localization in control GFP-KD (filled bar) or RAD21-KD SNU16 cells (unfilled bar) by unbiased automatic quantification (39). Data are presented as averages ± SD of individual cells (n = 100). *P < 0.01; Student's t test.