Figure 6.
ELDR interferes with the progression of DNA replication forks. (A) Representative image of colocalization of ELDR (red) and PCNA (green) in nuclear punctate structures using smFISH for ELDR and immunofluorescence with an anti-PCNA antibody and DAPI staining (blue). Scale bars, 5 μm. (B) Representative image of colocalization of ELDR (red) and MCM5 (green) in nuclear punctate structures using smFISH for ELDR and immunofluorescence with an anti-MCM5 antibody and DAPI staining (blue). Scale bar, 10 μm. (C) Spot detection using the IMARIS software for 3D reconstructions from immunofluorescence images for MCM5 and smFISH for ELDR detection. Spots represent individual signals for MCM5 (green) or ELDR (orange), respectively, and for MCM5-ELDR complexes (blue/purple). The cutoff for colocalization was 240 nm. Scale bar, 1 μm. Controls: representative 3D reconstruction of nuclei stained with an immunoglobulin G (IgG) control antibody and with human ELDR or murine ELDR (mELDR) probes for the smFISH. (D) Quantification of the percentage of cells among all analyzed cells with MCM5-ELDR complexes (ie, colocalized MCM5-ELDR spots per cell). Cell numbers were n = 94 for mELDR + IgG control, n = 63 cells for ELDR + IgG control, n = 109 for ELDR+MCM5. (E) Among cells with ELDR-MCM5 complexes, the number of colocalized spots per cell and the percentage of MCM5 colocalized with and without ELDR were quantified. (F-G) Relative EdU intensity measured by flow cytometry in S phase cells (see gating in Figure 2E) in THP-1 ELDR-overexpressing ELDR-C1 and ELDR-C3 cell lines compared with control cell lines EV1 and EV2 or in Mono-Mac-1 ELDR-overexpressing ELDR-C4 and ELDR-C7 cell lines compared with control cell lines EV2 and EV3, cultured in HS-5 conditioned media. (H) Left panel: schema of DNA fiber assay. Labeling scheme: CIdU (red) was incorporated as the first analog, followed by IdU (green), incorporated as the second analog at indicated time points. Right panel: HU was added to completely stop fork progression for indicated time point. (I) DNA fiber assay with same incubation times (30 minutes) for IdU and CIdU labeling with or without subsequent HU treatment. Plots of IdU/CIdU ratios and IdU or CIdU track lengths for the indicated THP-1 ELDR-overexpressing cell lines (ELDR-C1, ELDR-C3) and controls (EV1, EV2). Experiments were performed in 2 to 3 biological replicates and at least 150 to 200 dually labeled fibers were measured for each condition. Only bicolor fibers were analyzed, that is, forks that progressed normally during the first labeling period. (J) Schema of symmetric and asymmetric forks. In contrary to symmetric fork progression (A/B ≈ 1), both sides (A and B) of the origin will not progress at the same rate (A/B ≠ 1) in asymmetric fork resulting in fork stalling. (K) Quantification of frequency of asymmetric DNA replication forks (in percent). Total number of forks counted: 30 to 40 per sample. n = 3 biological replicates. Data are presented as mean + SD (panels E, K); ± SD (panel I). ∗∗∗P < .001; ∗∗∗∗P < .0001. P values were calculated using Kruskal-Wallis test (panel I); 1-way ANOVA with Tukey's multiple comparisons test (K). ns, not significant; HU, hydroxyurea.

ELDR interferes with the progression of DNA replication forks. (A) Representative image of colocalization of ELDR (red) and PCNA (green) in nuclear punctate structures using smFISH for ELDR and immunofluorescence with an anti-PCNA antibody and DAPI staining (blue). Scale bars, 5 μm. (B) Representative image of colocalization of ELDR (red) and MCM5 (green) in nuclear punctate structures using smFISH for ELDR and immunofluorescence with an anti-MCM5 antibody and DAPI staining (blue). Scale bar, 10 μm. (C) Spot detection using the IMARIS software for 3D reconstructions from immunofluorescence images for MCM5 and smFISH for ELDR detection. Spots represent individual signals for MCM5 (green) or ELDR (orange), respectively, and for MCM5-ELDR complexes (blue/purple). The cutoff for colocalization was 240 nm. Scale bar, 1 μm. Controls: representative 3D reconstruction of nuclei stained with an immunoglobulin G (IgG) control antibody and with human ELDR or murine ELDR (mELDR) probes for the smFISH. (D) Quantification of the percentage of cells among all analyzed cells with MCM5-ELDR complexes (ie, colocalized MCM5-ELDR spots per cell). Cell numbers were n = 94 for mELDR + IgG control, n = 63 cells for ELDR + IgG control, n = 109 for ELDR+MCM5. (E) Among cells with ELDR-MCM5 complexes, the number of colocalized spots per cell and the percentage of MCM5 colocalized with and without ELDR were quantified. (F-G) Relative EdU intensity measured by flow cytometry in S phase cells (see gating in Figure 2E) in THP-1 ELDR-overexpressing ELDR-C1 and ELDR-C3 cell lines compared with control cell lines EV1 and EV2 or in Mono-Mac-1 ELDR-overexpressing ELDR-C4 and ELDR-C7 cell lines compared with control cell lines EV2 and EV3, cultured in HS-5 conditioned media. (H) Left panel: schema of DNA fiber assay. Labeling scheme: CIdU (red) was incorporated as the first analog, followed by IdU (green), incorporated as the second analog at indicated time points. Right panel: HU was added to completely stop fork progression for indicated time point. (I) DNA fiber assay with same incubation times (30 minutes) for IdU and CIdU labeling with or without subsequent HU treatment. Plots of IdU/CIdU ratios and IdU or CIdU track lengths for the indicated THP-1 ELDR-overexpressing cell lines (ELDR-C1, ELDR-C3) and controls (EV1, EV2). Experiments were performed in 2 to 3 biological replicates and at least 150 to 200 dually labeled fibers were measured for each condition. Only bicolor fibers were analyzed, that is, forks that progressed normally during the first labeling period. (J) Schema of symmetric and asymmetric forks. In contrary to symmetric fork progression (A/B ≈ 1), both sides (A and B) of the origin will not progress at the same rate (A/B ≠ 1) in asymmetric fork resulting in fork stalling. (K) Quantification of frequency of asymmetric DNA replication forks (in percent). Total number of forks counted: 30 to 40 per sample. n = 3 biological replicates. Data are presented as mean + SD (panels E, K); ± SD (panel I). ∗∗∗P < .001; ∗∗∗∗P < .0001. P values were calculated using Kruskal-Wallis test (panel I); 1-way ANOVA with Tukey's multiple comparisons test (K). ns, not significant; HU, hydroxyurea.

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