EMBO/SourceData huggingface dataset:
Figure 2(A-C) 3D rendering of Z-stack confocal image showing Isolectin B4-stained laser-burn with FITC-dextran-labeled CNV and IBA-1-stained MPs.(D) Representative FACS plots of retinas and sclera-choroid-RPE-cell complexes from Regular-chow Diet (RD)-fed mice without and 3, 7 and 14 days after laser-burn. (E) Quantification of MPs (Ly6G -, F4/80+, CD11b+) at p3, p7 and p14; fold naïve (no burn); n= 5(no burn), 5(p3), 6(p7), 5(p14); CI 95%. (F) Representative FACS plots and (G) quantification of microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hiCD45lo) fold naïve; n= 5(no burn), 4(p3), 6(p7), 5(p14); CI 95%.(H) Representative FACS plots of retinas and sclera-choroid-RPE-cell complexes from RD- and HFD-fed mice, with vehicle or antibiotic treatment at p7. (I) Quantification of MPs at p7 fold RD; n= 6(RD), 4(HFD), 3(HFD+AB), 4(RD+AB); CI 95%. (J) Representative FACS plots and (K) quantification of microglia (Ly6G-, F4/80+, CD11b+, CX3CR1hiCD45lo) fold RD; n= 5(RD), 5(HFD), 3(HFD+AB), 4(RD+AB); CI 95%.(L) Representative confocal images of IBA-1-stained MPs on choroidal flat mounts from RD- and HFD-fed mice, treated with vehicle or with neomycin. Examples of labeled macrophages (white dots) are presented in side-panels. (M) Total number of MPs around laser impact area; n= 7(RD), 9(HFD), 4(HFD+AB), 7(RD+AB), with 23(RD), 29(HFD), 13(HFD+AB), 23(RD+AB) burns total; CI 99%.
Figure 3(A) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in Regular-chow Diet (RD)- and high-fat diet (HFD)-fed mice, treated with vehicle or with antibiotic treatment; n= 6(RD), 5(HFD), 5(HFD+AB), 5(RD+AB); CI 99%.(B) Activation of PRRs induced by stimulation with serum isolated from mice fed RD or HFD for 7 weeks and receiving vehicle or neomycin for 3 weeks; fold RD; n=3 for all groups; CI 99%.(C) Serum cytokine profiles determined by Bio-Plex assay (Bio-Rad) of IL-1β, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; IL-6, n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB), CI 99.9%; TNF-α n=11 for all groups, CI 99%; IL-17A n= 11(RD), 12(HFD), 12(HFD+AB), 11(RD+AB); CI 95%; and IFN-γ; n= 12 for all groups; CI 95%.(D) mRNA expression of Il-1β; n= 5(RD), 7(HFD), 7(HFD+AB), 3(RD+AB); CI 95%, Il-6; n= 5(RD), 5(HFD), 4(HFD+AB), 4(RD+AB); CI 95%, Tnf-α; n= 6(RD), 9(HFD), 4(HFD+AB), 5(RD+AB); CI 95% and Vegf-a; n= 6(RD), 5(HFD), 5(HFD+AB), 6(RD+AB); CI 95% in choroids.
Figure 4(C) Weight gain and (D) area under the curve of percentage weight gain of RD x RDT, HFD x RDT and HFD x HFDT mice; n= 13(RDxRDT), 12(HFDxHFDT), 14(HFDxRDT); CI 99%.(E) Representative circle charts of relative abundance of bacterial phyla in gut microbiota of RD-fed mice receiving a RD transfer and HFD-fed mice receiving a HFD or RD transfer(F) relative proportion per experimental group of different phyla; n= 5(RDxRDT), 5(HFDxHFDT), 4(HFDxRDT); Bacteroidetes; CI 99%, Firmicutes; CI 95%, Proteobacteria; CI 95%, Actinobacteria and Spirochaetes.(G) Compilation of compressed Z-stack confocal images of FITC-dextran-labeled CNV and single-plane confocal images of isolectin B4-stained choroidal flat mounts from RD x RDT, HFD x RDT and HFD x HFDT mice. (H) Quantification of area of FITC-dextran-labeled CNV over area of isolectin B4-stained laser impact area fold RD x RDT; n= 6(RDxRDT), 9(HFDxHFDT), 9(HFDxRDT), with 15(RDxRDT), 21(HFDxHFDT), 16(HFDxRDT) burns total; CI 95%.(I) Concentration of gut absorbed Evans Blue in serum 24 hours after oral administration in RD- and HFD-fed mice, after RD or HFD microbiotal transplant; n= 3(RDxRDT), 3(HFDxHFDT), 4(HFDxRDT); CI 95%.(J) mRNA expression of Il-1β; n= 5(RDxRDT), 4(HFDxHFDT), 5(HFDxRDT), Il-6; n= 5(RDxRDT), 4(HFDxHFDT), 4(HFDxRDT); CI 99%, Tnf-α; n= 5(RDxRDT), 4(HFDxHFDT), 4(HFDxRDT); CI 95% and Vegf-a; n= 5(RDxRDT), 4(HFDxHFDT), 5(HFDxRDT); CI 95% in choroids.
Figure 1(A) PKH26 (PE)-labelled RM1 (V500/eCFP) cells were injected intracardiac (IC) into C57BL/6 mice and FACS-isolated from bones with evident tumor burden (8 individual mice across 5 independent experiments) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA-seq. Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH+ (PE/V500) and PKH- (V500) RM1 cells and bone marrow cells (grey).(B) goana gene ontology (GO) analysis (limma) of all DE genes enriched in proliferating (PKH-; n = 32) cells compared to dormant cells (PKH+; n = 28). Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.(C) goana GO analysis (limma) of all DE genes uniquely enriched in PKH+ compared to PKH- cells. Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.(E) Heatmap of relative BASiCS-derived log2(denoised counts + 1) of type I IRGs (upregulated > 2-fold in the INTERFEROME in at least one dataset) that are differentially expressed between all dormant and active cells with no residual over-dispersion. Mice IDs for each sample are indicated, along with type I IRGs that are identified as such in at least 30 INTERFEROME datasets. Genes are displayed if detected in at least 10 samples per population. Samples with zero counts for individual genes are represented in white.(F) Dot plot of BASiCS-derived log2(denoised counts + 1) of key DE IRGs expressed in > 5 dormant cells associated with immune-activatory processes (among one of the most enriched types of biological pathway determined by GO analyses) present for all single cells ranked by ExpLogFC (Fig. 1c). Open circles are zero counts. Bars indicate mean expression.
Figure 2(A Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone-derived (BD) cells compared to parental RM1 and lung metastases. Tumor-intrinsic type I IFN signaling is suppressed in proliferating bone lesions (n = 1) compared to parental RM1 (n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the -log10FDR; false discovery rate). Color indicates mean log2fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN-α response gene set in genes suppressed in bone metastases, using the signed log-likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set;(C) log2FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN-α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. p values represented as * < 0.05, ** < 0.005 and *** < 0.0005; GLM by edgeR.(E) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases (n = 1) compared to lung metastases (n = 1) and parental cells (n = 2).(F) qRT-PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung) and naïve bone marrow (BM) (n = 3 mice per group). p values represented as ** < 0.005 and *** < 0.0005 (Student's t test).(G) qRT-PCR of Irf7 and Irf9 downregulation in parental RM1 cells, RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 deficient (-/-) mice, with naïve BM from WT and Ifnar1 (-/-) animals for reference. (n = 3 mice per group; n = 1 for -/- BM control). p values represented as * < 0.05 and ** < 0.005 (Student's t test).
Figure 3(A) Stability of Irf7 and Irf9 mRNA suppression by qRT-PCR in ex vivo bone-derived cells (RM1 BD Irf-; n = 7) in culture compared to a bone-derived line that showed initial loss during early passage (RM1 BD REV (EP); n = 3) then reverted to parental expression levels at late passage (RM1 BD REV (LP); n = 4) compared to parental RM1 (n = 3).(B) ELISA of IFN-α production by RM1 parental (n = 3), RM1 bone-derived Irf low (RM1 BD Irf-; n = 4) and RM1 BD REV cells (n = 3) subsequent to poly I:C stimulation.(C) qRT-PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf- cells ± 48 h treatment with MS275 (1 μM) (n = 7-9).(e) qRT-PCR analysis of Irf7 and Irf9 expression in parental RM1 cells (n = 4) 48 h post-contact culture with naïve BM (n = 6).(F) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co-culture with naïve BM under contact (non-transwell; NT) and transwell (0.4 μm filters that prevent cell contact) conditions (n = 6-8 per condition).(G) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co-culture with naïve BM ± MS275 (1 μM) (n = 3-6 per condition).
Figure 4(A) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.0043 by log-rank (Mantel-Cox) test).(C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4).(D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test).(E) Bone metastasis-free survival in Ifnar1 -/- mice harboring RM1 BD Irf- BV and RM1 BD Irf7 OE tumors (n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F).(F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm.(G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits.(J) FACS analysis of IFN-γ+ and TNF-α+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation by RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE cells (n = 6 per condition/group).(K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.
Figure 5(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).(B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3).(C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1)FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown.(E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test).(F) Mean total tumor burden in bone (femurs, tibias, spine and humerus) at endpoint by bioluminescent intensity score (log2 p/sec/cm2/sr) with values < 4.0x104 representing zero burden (n = 24 bones per group; 6 mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Figure S5E).
Figure 6(A, FACS analysis of peripheral blood (PB) T and NK lymphocyte representation and activation statusB) FACS analysis of FoxP3+ CD4+ and CD4+ effector memory T cell status at days 4 and 10 post-IC tumor cell inoculation across treatment settings (n = 4-6 per group).(C, Bioluminescent imaging (C) of a combination group mouse with tumor clearance from day 7 to 41 (full experimental cohort shown in Appendix Fig S5E)D) post-IC inoculation with associated specific CD8+ memory T cell response. This is represented by FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation upon re-stimulation with RM1 BD Irf- cells and MS275, poly I:C or combination treatments in vitro, with NAC and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right.
Figure 7Compositional analysis of RNA-seq data (FDR < 0.05; GLM by edgeR) from bone metastases (n = 9) and primary tumors (n = 12) revealed (A) immune cells and (B) tumor cells were relative (% of total) across all samples, with a mean tumor purity >85% as determined by ARMET. Median shown. Upper and low box hinges denote first and third quartiles. Upper and lower whiskers mark the values ± 1.5IQR from the hinge. Data points beyond the whiskers are shown individually. p values not significant by Wilcoxon rank sum test (R).Hallmark gene set responses associated with bone metastasis by camera analysis shows (C) enrichment of type I (IFN-α) IFN responses in suppressed genes (barcode plot).(D) Heatmap of type I IFN DE IRGs (normalized log2CPM; counts per million) significantly suppressed in bone metastases (n = 9) compared to primary (n = 12) and benign (n = 3) tumors, grouped by type I IFN specific, and type II and III IFN co-regulation status. Scale truncated at ± 5 relative log2CPM. GLM by edgeR.(E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C.(F) Heatmap of HLA genes (normalized log2CPM) suppressed (p <0.05; FDR <0.05) in bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). Scale truncated at ±5 relative log2CPM. GLM by edgeR.(G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U.(H) Kaplan Meier Curve of human prostate (n = 499 from 498 TCGA samples) biochemical recurrence based on primary tumor alterations in a core 8-IRG signature (Appendix Fig S7D). Patient groups stratified by mRNA z-Score (RNA-seq V2 RSEM) ± 1.5. Hazard ratio 1.674 (95% confidence interval (CI) 2.707 to 1.035); *p = 0.03 by log-rank test.
Figure 1Viability of single and double mutants of RNAPII and rad52Δ, mre11Δ and cdc44‐8. Tenfold serial dilutions of double mutants obtained by genetic crosses were tested for growth in plain SC medium or supplemented with 10 mM HU or 0.005% MMS.Tetrad analysis of rpb1‐S751F, rad52Δ and mre11Δ crosses. Δ indicates double mutants that fail to grow.Tetrad analysis of rpb1‐S751F crossed by pol32Δ and rad51Δ. Δ indicates triple mutants that fail to grow.HU and MMS sensitivity of different mutant combinations of rpb1‐1, rpb1‐S751F, pol32Δ and rad51Δ.
Figure 2Western blot against the phosphorylated form of histone H2A in the presence of 100 mM HU in asynchronously growing WT and RNAPII mutant cells. Ponceau staining is shown as a loading control.Rad52 foci formation at 30 and 37°C.Recombination frequencies between inverted repeats in WT and RNAPII mutants measured with the plasmid‐born pTINV system. Recombinants were selected as Leu+.Recombination frequencies using the direct‐repeat chromosomal system leu2‐k::ADE2‐URA3::leu2‐k in WT and RNAPII mutants. Deletion events were detected and quantified in media containing 5‐fluorotic acid (FOA) and could be directly visualized as red sectors.Recombination frequencies between plasmid‐born direct repeats in WT and RNAPII mutants. The frequency of Leu+ recombination was determined in the systems L‐lacZ and GL‐lacZ. Recombination frequencies are plotted as a function of the transcription levels. Low transcription refers to the GL‐lacZ systems in strains cultured in 2% glucose; medium refers to L‐lacZ in 2% glucose, and high to GL‐lacZ in 2% galactose.Mitotic stability of centromeric plasmid pGAL‐lacZ in RNAPII mutants. Stability was analyzed in galactose after 23 generations. A small diagram of each system (not drawn to scale) is shown.
Figure 3Recombination frequencies measured with the pTINV system in WT, rpb1‐1, mre11Δ and rpb1‐1 mre11Δ.Rad52 focus formation in asynchronously growing cells at 30°C.Viability after different times in 10 mM HU. The same cultures without HU were used as a control. The average values and standard deviations from three independent experiments are shown.Rad52 focus formation in asynchronously growing cells expressing normal or high levels of RNH1.
Figure 4FACS analysis of cell cycle progression in rpb mutants. Cells were synchronized in G1 with α‐factor and monitored at different times after release in the presence of 40 mM HU.Slowdown of RF progression through a chromosomal region. 2D gel electrophoresis of replication intermediates of 4.3‐kb PstI DNA fragments from WT and RNAPII mutants in the presence of 40 mM HU at different times after G1 release. A black arrow indicates accumulation of long Y molecules, and a white one indicates the specific accumulation of X molecules.
Figure 5Analysis of replication by BrdU incorporation. Cells were synchronized in G1 with α‐factor and released in the presence of BrdU. Samples were taken at the indicated times and processed to measure BrdU incorporation by ChIP analysis. Replication was analyzed in two DNA regions, one covering the early origin ARS508 and the SPF1 gene and the other the late origin ARS603 and the YFL034W gene.DNA combing analysis of replication. Graphs indicate the distribution of BrdU track length (kb) at 60 and 120 min after α‐factor release in the presence of 40 mM HU, the replication speed (kb/min) determined with the two time points analyzed and the inter‐origin distance (kb) at 60 min after release. The median test was applied.
Figure 6Dephosphorylation of Rad53 after exposure to HU. WT, rpb1‐1 and rpb1‐S751F cells were synchronized in G1 with α‐factor. After G1 release, cells were exposed to 100 mM HU for 1 h, washed to remove HU and cultured in HU‐free medium for the indicated periods. Western blot against Rad53, signal quantification (ratio Rad53‐P/total Rad53 protein) and FACS profiles are shown.DNA repair of MMS damage. Chromosome VII species revealed by hybridization with ADE5,7 probes in pulsed‐field gel electrophoresis (PFGE). Asynchronously growing WT and rpb1 cells were exposed to 0.1% MMS for 30 min, washed to remove MMS and cultured in MMS‐free medium for the indicated periods to allow repair. Repair was calculated as the percentage of FLC appearing after MMS treatment. The FLC signal was quantified with respect to the total signal of each lane. Data show the mean and SD of 3 independent experiments. NLC, nonlinear chromosome. FLC, full‐length linear chromosome. CF, chromosome fragment resulting from DNA breaks.
Figure 7Retention of RNAPII in the actively transcribed PMA1 gene in RNAPII mutants. ChIP analysis of RNAPII in asynchronously growing WT, rpb1‐1, rpb1‐S751F, rpb2‐10 and rpb9Δ cells at the PMA1 gene. The scheme of the gene and the PCR‐amplified fragments are shown. Recruitment values were calculated from the amount of DNA present in each region, normalizing IP/input ratios, and relative to the amount of DNA of the intergenic region. Error bars indicate the standard deviation (SD).Northern blot analysis of the expression of PMA1 gene. RNA was isolated from mid‐log phase cultures grown in YPD‐rich medium. As 32P‐labeled DNA probes, an internal 776‐bp PMA1 fragment and an internal 513‐bp SCR1 fragment obtained by PCR were used. Bars indicate the SD of 2 independent experiments.
Figure 8Analysis of Rrm3 recruitment in WT and rpb1‐1. Rrm3 IP histogram bars in the y‐axis show the average signal log2 ratio of loci enriched in the immunoprecipitated fraction along the indicated regions in the x‐axis. When the ratio fulfills the P‐value criteria (P 0.01) it is shown in red.Composite profile of Rrm3 occupancy across the average ORF plotted as Rrm3 percentage of significant ChIP hits per segment. Each gene was divided into 10 equivalent segments from the start and end coordinates (segments 2-10). Two additional segments of the same size were considered upstream (5′) and downstream (3′) of each ORF (segments 1 and 12, respectively). The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.Composite profile of Rrm3 occupancy across the average ARS plotted as Rrm3 percentage of significant ChIP hits per segment. Each ARS was divided into 11 equivalent segments from the start and end coordinates. The average signal log2 ratio for Rrm3 significant hits that map on each segment was plotted.
Figure 1(C) PCR analysis of a pool of transfected cells after treatment with Cas9-D10A 'nickase' and the indicated combination of gRNAs The ~200 bp product was centred around the start of exon 1 of the SLD5 gene. Efficient cutting of both template strands by Cas9-nickase was indicated by the production of a smeary PCR product (gRNAs 2+3), reflecting DNA repair that mostly produced small deletions around the nicking site The combination of gRNAs 1+3 did not produce a smeary PCR, due to a polymorphism within the PAM motif of the genomic locus(F) Immunoblot indicating successful tagging of both alleles of SLD5 with TAP or GFP.(G) Asynchronous cultures of the indicated cells were used to generate cell extracts, which were then incubated with agarose beads coupled to anti-GFP nanobodies.(H) Extracts of TAP-SLD5 mouse ES cells were incubated plus or minus DNase for 30 minutes at 4 °C, before immunoprecipitation of TAP-SLD5.(I) TAP-SLD5 cells were synchronised as indicated DNA content was monitored throughout the experiment by flow cytometry.Samples were taken at the indicated times and used to isolate TAP-SLD5 by immunoprecipitation on IgG beads. The indicated proteins were monitored by immunoblotting. For reasons of space, samples A, S, M and 1-6 were resolved in a separate gel to samples 7-8.
Figure 2(A) Cells were treated with 5 µM CB-5083 as indicated (p97i = p97 inhibitor). Extracts were then incubated with IgG beads to isolate the GINS complex via TAP-SLD5 and the indicated factors were monitored by immunoblotting. Accumulation of the CMG helicase upon inhibition of p97 was reflected by the increased association of GINS with CDC45 and MCM2-7.(B) Immunostaining of HP1 protein in fixed mouse ES cells that were stained with Hoechst to reveal condensed patches of heterochromatic DNA (examples marked by arrows; colocalization of HP1 and condensed DNA in 83 % of cells, n = 103).(C) Time-lapse analysis of mouse ES cells expressing GFP-SLD5 and mCherry-PCNA Arrows indicate co-localisation on heterochromatic patches, which was observed in 14 % asynchronous cells (n = 238). GFP-SLD5 appeared and disappeared with similar kinetics to mCherry-PCNA (100 % cells, n = 101).(D) GFP-SLD5 mouse ES cells were treated with p97i as indicated and then fixed and stained with Hoechst. Arrows indicate accumulation of GFP-SLD5 on constitutive heterochromatin (53 % cells, n = 125).(E) Immunofluorescence of the replisome components CLASPIN (i) and CTF4 (ii) in GFP-SLD5 cells treated with p97i. Arrows indicate co-localisation of GFP-SLD5 patches with CLASPIN (95 % cells, n = 107) or CTF4 (91 % cells, n = 45). Scale bars in all panels correspond to 10 µm.