EMBO/SourceData huggingface dataset:
Figure 4(B, Protein expression in liver tissue of cleaved and mature forms of Casp1 (p20), IL-1β and TNF-α (17kDa) (n=3 mice per genotype for Western blots).C) Protein expression in liver tissue of cleaved and mature forms of Casp1 (p20), IL-1β and TNF-α (17kDa) Data information: n≥6 mice per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats
Figure 5(A) Immunohistochemical staining of F4/80, a marker for resident macrophages, and mRNA expression of F4/80 in liver tissue. Data information: Representative images shown, bar indicates 250 μM for F4/80 n≥6 mice per genotype, significance determined by unpaired t test (** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats(B) Immunofluorescence staining of CLEC4F, a specific marker of Kupffer cells, and gene expression of CLEC4F in liver tissue. Data information: Representative images shown, bar indicates 100 μM for CLEC4F, n≥6 mice per genotype, significance determined by unpaired t test (** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats(C) Immunohistochemical staining of Ly6C, a marker for pro-inflammatory macrophages, in liver tissue. Data information: Representative images shown, bar indicates 250 μM for Ly6C, n≥6 mice per genotype, significance determined by unpaired t test (** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats
Figure 6(A) Collagen deposition assessed by Sirius red staining Data information: Representative images shown, bar indicates 250 μM, n≥6 per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats(C) Protein expression of collagen 1a in whole liver lysate. n=3 Data information: n≥6 per genotype, significance determined by unpaired t test (* p < 0.05; ** p < 0.01; *** p < 0.001), data are expressed as mean +/− SD, dots represent individual biological replicats
Figure 2A Immunoblot analysis with the indicated antibodies of immunoprecipitates (IP) prepared from the SSU fraction of eIF3D-V5/eIF4E-FLAG dKI HEK293T cells (eIF3D/4E dKI) or wild-type (WT) cells with antibodies to the epitope tags. The SSU fraction (input) was also analyzed directly. Cells were fixed with formalin, cell lysates were treated with RNase, and the SSU and RS fractions were separated by sucrose density gradient centrifugation before immunopurification. Molecular sizes are indicated in kilodaltons.B, C Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the SSU fraction of eIF4A1-FLAG KI HEK293T cells (eIF4A1 KI) (B) or ASCC3-FLAG KI HEK293T cells (ASCC3 KI) (C).Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the RS fraction of RPL7A-HA KI HEK293T cells (RPL7A KI) (F).
Figure 4A RNA-seq results for eIF4A1, eIF4A2, ASCC2, and ASCC3 in HEK293T cells transfected with siRNAs specific for eIF4A1 and eIF4A2 (eIF4A1/A2 dKD #1 or #2) or for ASCC3 (ASCC3 KD #1 or #2) relative to cells transfected with a control siRNA. The dashed line indicates a fold change of 1.B Immunoblot analysis of the siRNA-transfected cells with the indicated antibodies.Volcano plots for differential TE (D) The changes in TE and SRO for eIF4A1/A2 dKD #1 and #2 and for ASCC3 KD #1 and #2 HEK293T cells compared with control cells were analyzed with RiboDiff. The gray dashed lines indicate an adjusted P value of 0.05. P values were calculated by chi-square test and adjusted by Benjamini-Hochberg method.
Figure 5D, E Cumulative fraction for fold change in TE in eIF4A1/A2 dKD or ASCC3 KD cells relative to control cells. Results are shown for all coding transcripts and for transcripts with a decreased (D) or increased (E) SRO. The P values were calculated with the Mann-Whitney U test.
Figure 6D Cumulative fraction for fold change in TE in eIF4A1/A2 dKD or ASCC3 KD cells relative to control cells. Results are shown for all coding transcripts and skewed transcripts. The P values were calculated with the Mann-Whitney U test.E Number of transcripts showing significant down-regulation of TE among skewed transcripts in eIF4A1/A2 dKD and ASCC3 KD cells.
Figure 7Luciferase assays for RADX, DNAJB6, and LDHA constructs (B) in control and ASCC3-depleted HEK293T cells. The results of the luciferase assays (right) and schematic representations of the constructs (left) are shown. Rluc, Renilla luciferase; Fluc, firefly luciferase. Assay data are means ± s.d. for three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, NS (two-sided Student's t test).Luciferase assays for corresponding deletion mutants of the RADX and DNAJB6 constructs (C) in control and ASCC3-depleted HEK293T cells. The results of the luciferase assays (right) and schematic representations of the constructs (left) are shown. The deleted regions of the RADX and DNAJB6 constructs are indicated by dashed white boxes. Rluc, Renilla luciferase; Fluc, firefly luciferase. Assay data are means ± s.d. for three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001
Figure 1(A) CD127+ and CD127- cells from Lin-CD56- population after gating on lymphoid, singlet, live, CD45+ cells of PBMCs. Lineage (Lin) markers include antibodies against: CD3, CD4, TCRαβ, TCRγδ, CD19, CD20, CD22, CD34, FcεRIα, CD11c, CD303, CD123, CD1a, and CD14.(C) Normalized counts of Lin-CD56-CD127+ (red) and Lin-CD56-CD127- (blue) cells related genes from (B) by DESeq2 (n=5).
Figure 2(A) Heatmap of differentially expressed genes by RNA-Seq, sorted CD56hiNK, CD56dimNK, CD56-NK cells and ILCs, from PBMCs of 4 donors (log2 fold change >1, p<0.01 determined by DESeq2(H) ATAC-Seq analysis of sorted CD56hiNK, CD56dimNK, CD56-NK cells and ILCs at indicated gene loci (representative of two donors).
Figure 3C) Representative genes for NK cell and ILC clusters. All data were generated using blood from healthy HIV-1-negative donors.(G) the expression of genes that are enriched by NK cells and/or ILCs along the pseudotime trajectory. All data were generated using blood from healthy HIV-1-negative donors.
Figure 5(A) PBMCs were stimulated with PMA and ionomycin for 3 hrs and then intracellular AREG was detected within the different subsets of NK cells and ILCs by flow cytometry.(B) PBMCs were cultured in MACS NK medium at 37 °C in 5% CO2, 50 ng/ml IL-2, 50 ng/ml IL-15, 10 ng/ml IL-12 or 50 ng/ml IL-18 for 5 days, then AREG, IFN-γ or TNF-α from different subsets of NK cells and ILCs were detected.(F PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of AREG+NK cells (F) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.G) PBMCs were treated with CHIR99021 (10 uM) in RPMI 1640 at 37 °C in 5% CO2 for 48 hrs, then stimulated with PMA+ionomycin. The percentage of ILCs (G) (n=15) are shown. two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.(H) As in (F), PBMCs were additionally treated with CHIR99021 and TGF-β1 (50 ng/ml) before AREG+NK cells were detected by flow cytometry (n=8). two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.(I) AREG+NK cells from control or RUNX3 knockout groups were detected after IL-12+IL-15+IL-18 stimulation in MACS NK medium at 37°C in 5% CO2 for 16 hrs (n= two-tailed paired t-test. All data were generated using blood from healthy HIV-1-negative donors.
Figure 6(A PBMCs from HIV-1- (n=20), HIV-1+ viremic (n=20), HIV-1+ ART suppressed (n=19), and HIV-1+ spontaneous controllers (n=20) were stimulated with PMA and ionomycin in RPMI 1640 at 37 °C in 5% CO2 for 3 hrs. The percentage (A) of AREG+ cells from are shown for the indicated populations. Mann-Whitney test. ns, not significant.(C-F) Correlation of the percentage of AREG+ cells in CD56- (C), CD56dim (D), CD56hi (E) or total NK cells (F), from (A), with the percentage of ILCs. Spearman correlation
Figure 7(A) Percentage of ILCs among Lin-CD56- cells from HIV-1-negative (n=40), HIV-1+ viremic (n=40), HIV-1+ ART suppressed (n=37), and HIV-1+ spontaneous controllers (n=40).(E) Percentage of CD56-NK cells among total NK cells from PBMCs and CD4-PBMCs after culture in RPMI 1640 at 37°C in 5% CO2 for 5 days (n=7).(F) Percentage of CD56-NK cells bearing the indicated proteins by flow cytometry, after detection in fresh PBMCs or after 5 days of culturing CD4+T cell-depleted PBMCs (n=4).(G) CD4+T cell number (counts/mm3) from the indicated groups (n=40 for each group).
Figure 8(A) CD56dim and CD56- NK cells were assessed by flow cytometry after the indicated cell populations were cultured for 5 days: total PBMCs (n=7 blood donors), PBMCs depleted of CD4+T cells (n=7 blood donors), NK cells mixed with autologous CD4+T cells (n=3 blood donors), and PBMCs depleted of CD4+T cells but supplemented with IL-2 (n=7 blood donors).(B) Percentage of CD56-NK cells among total NK cells from CD4-PBMCs cultured as in (A), in the presence or absence of IL-2 (10 ng/ml) or IL-2 neutralizing antibody (4 ug/ml) (n=7). (C) Percentage of CD56-NK cells from PBMCs or CD4-PBMCs cultured as in (A) in the presence or absence of IL-2 (10 ng/ml), or IL-15 (10 ng/ml) (n=8). (D) Percentage of CD56-NK cells among total NK cells in PBMCs after culturing as in (A) with isotype control or IL-2 neutralizing antibody (4 ug/ml) (n=10).(E, F) Percentage of CD56-NK (E) or IFN-γ+ NK (F) cells among total NK cells after culturing PBMCs as in (A) with TGF-β1 (50 ng/ml) or TGF-β1 (50 ng/ml) + IL-2 (20 ng/ml) (n=8).(I, J) NSG (n=15) or human IL-15 transgenic NSG (NSG-huIL-15) mice (n=15) were reconstituted with human CD34+ hematopoietic stem cells for 6 weeks. Blood, spleen, and liver were harvested for detection of CD56 (I) or IFN-γ production after 16 hrs stimulation with IL-12, IL-15, and IL-18 (J).(K-L) Plasma concentration of IL-2 (HIV-1- (n=12), viremic (n=12), ART suppressed (n=13), controllers (n=14)) (K), TGF-β1 (HIV-1- (n=20), viremic (n=19), ART suppressed (n=20), controllers (n=20)) (L) and IL-15 (HIV-1- (n=19), viremic (n=19), ART suppressed (n=20), controllers (n=20)) (M), detected by Luminex.
Figure 9(C) Log2 fold change of mTOR signaling-related genes, showing cells stimulated with IL-12, IL-15, and IL-18 versus unstimulated CD56- or CD56dim NK cells. ns, not significant. All data were generated using blood from healthy HIV-1-negative donors.
Figure 10(A) PBMCs were cultured at 37 °C in 5% CO2 with or without IL-2 (50 ng/ml) for 5 days, the phosphorylation of mTOR on Ser2448, AKT on Ser473, 4EBP1 on Thr36 and Thr45, and S6 on Ser235 and Ser236, and the surface CD71 from NK cells (Lin-TBX21+) were detected by flow cytometry (n=8). (B) As in (A), PBMCs or CD4-PBMCs were cultured for 5 days, the indicated targets from NK cells were detected (n=8). (C) As in (A), PBMCs were cultured with or without IL-15 (50 ng/ml) for 5 days, the indicated targets were detected (n=8). All data were generated using blood from healthy HIV-1-negative donors.(E, F) PBMCs or CD4-PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F, n=8) or Torin 1 (250 nM) (G, n=8), with or without IL-2 (10 ng/ml) or IL-15 (10 ng/ml), for 5 days. IFN-γ was detected in Lin-TBX21+ cells. All data were generated using blood from healthy HIV-1-negative donors.(G, H) PBMCs were cultured in the presence or absence of rapamycin (10 nM) (F) or Torin 1 (250 nM) combined with or without IL-2 (10 ng/ml) (G, n=8) or IL-15 (10 ng/ml) (H, n=8) for 5 days. The indicated targets from the Lin-TBX21+ population were detected as in (A). All data were generated using blood from healthy HIV-1-negative donors.
Figure 1A) Overview of immunofluorescence staining for centrosomes (TUBG1) and cilia (ARL13B) in control (Con) (left) and EPZ treated sections (right). Dotted lines mark either ventricular (bottom part) or pial (top part) surface of the tissue . Data information: Scale bars in all panels: 10 µm.B) Left: quantification of total centrosome numbers in control and EPZ treated sections in 100 µm. Right: number of centrosomes in ventricular zone (VZ), subventricular zone (SVZ), and cortical plate (CP) expressed as percentage of total per condition. C) Left: quantification of total cilia numbers in control and EPZ treated sections in 100 µm. Right: number of cilia in VZ, SVZ, and CP expressed as percentage of total per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.D) Overview of VZ showing TUBG1 and ARL13B immunofluorescence staining in control (top) and EPZ (bottom) condition. Squared parentheses indicate the ventricular and abventricular compartment. Dotted lines mark ventricular surface of tissue slide. Data information: Scale bars in all panels: 10 µm.E) Quantification of TUBG1- and (F) ARL13B-positive structures in ventricular and abventricular compartment expressed as percentage of the total number per condition. Data information: T-test, two-tails was performed for all quantifications, *p<0.05, ***p<0.001, error bars represent SD.
Figure 2A) Immunostaining of control and EPZ treated sections showing SOX2 and EOMES expression and EdU labelling. Scale bar: 20 µm. Dotted lines mark ventricular surface of the tissue. B) Quantification of SOX2-, EOMES-, EdU- positive cells, and co-labelled cells normalised to 100 μm of apical surface. T-test was performed, *p<0.05, **p<0.01, error bars represent SEM. Total n of independent experiments for EdU labelling: 9 (n=4 control samples, n=5 EPZ-treated samples). Total n of independent experiments for SOX2, EOMES, and double stainings: 6 (n=3 control samples, n=3 EPZ-treated samples).E) Immunofluorescence staining showing overview of CTIP2-positive cells in control and EPZ treated sections. Scale bars: 20 µm. Dotted lines mark either ventricular or pial surface of the tissue. F) Quantification of total CTIP2-positive cells, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). Error bars represent SEM.G) Immunofluorescence staining showing SOX2-, EOMES-, and CTIP2-positive cells in control and EPZ treated sections. Scale bars: 10 μm. Dotted lines mark the ventricular surface of the tissue. Magnifications on the right show exemplarily a CTIP2 positive cell (arrowhead) that is negative for SOX2 and positive for EOMES in EPZ treated condition. H) Quantification of CTIP2-positive cells in the VZ, normalised to 100 µm of apical surface. Total n of independent experiments: 6 (n=3 control samples, n=3 EPZ-treated samples). T-test was performed, **p<0.01, error bars represent SEM.I) In utero electroporation of cortical tissue of shRNA against DOT1L and control alongside a GFP expressing plasmid, followed by assessment of expression of CTIP2 and TUBB3 as markers for neuronal differentiation to study cell-autonomous and non-autonomous effects. Scale bars: 20 µm. Dotted lines mark either ventricular or pial surface of tissue slide. J) Quantification of CTIP2-, and TUBB3-positive cells in VZ, SVZ, IZ, and CP in control and shDot1l sections. Total n of independent experiments: 6 (n=3 control shRNA, n=3 shDot1l ). T-test was performed, *p<0.05, error bars represent SEM.
Figure 3A) Microinjected cells in the VZ (magenta) in sections treated with DMSO (Con) (upper panel) or EPZ (lower panel) showing Dx-A555 labelled cells after 24 h in slice culture. White arrow indicates the apical process contacting the ventricular surface. White arrowheads indicate the apical-directed process of delaminated cells. Scale bars: 10 µm. B) Immunofluorescence staining of microinjected sections showing the progeny of microinjected cells (a daughter cell pair) and expression of EOMES and TUBB3. White arrows indicate the apical-directed processes. Note that in the top panel the apical-directed process contacts the ventricular surface (dotted white line) (EOMES-, TUBB3-negative), while in the bottom panel it does not contact the ventricular surface, indicating that the cell has delaminated (EOMES-, TUBB3-positive). Scale bars MIP: 10 µm; insets: 5 µm. Data information: VZ - Ventricular Zone, SVZ - Subventricular Zone, CP - Cortical Plate. Scale bars in all main panels: 20 µm, and all insets: 5 µm.C) Quantification of microinjected cells found as single-cell or two-cells clusters, expressed as percentage of total Dx-A555-positive cells per condition. D) Quantification of microinjected cells with or without contact to the apical surface of the organotypic slice expressed as percentage of total Dx-A555-positive cells per condition. Total n of independent microinjection experiments: 6 (n=3 wild type embryos, n=3 in Tis21-GFP embryos). Total number of cells scored: Con, 156 cells; EPZ, 287 cells. E) EOMES-positive and EOMES-negative cells plotted as a percentage of total cells scored per condition. F) TUBB3-positive and TUBB3-negative cells in control and EPZ conditions plotted as a percentage of total cells scored per condition. Data information: Cell scoring based on TUBB3 or EOMES expression are from three independent experiments (n=3; Con: 70 cells, EPZ: 113 cells). Fisher's exact test was performed for all quantifications, ***p<0.001, error bars represent SEM. MIP - maximum intensity projection.
Figure 6C) Immunostaining showing ASNS-positive cells in the VZ, SVZ and CP of control and EPZ treated sections; and signal intensity for each condition. Scale bars: 50 µm. Dotted lines mark either ventricular or pial surface of the tissue .D) ChIP-seq and ATAC-seq tracks showing distribution of peaks for selected chromatin marks on the Asns locus for NPCs and cerebral cortex of E12.5 and E14.5 mice. Tracks for control and EPZ conditions are compared in NPCs. Indicated is the TSS of Asns.F) ChIP-qPCR analysis for DOT1L and IgG (n=7; biological replicates) at the TSS on Asns genomic locus in in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. **p<0.01. G) ChIP-qPCR analyses for EZH2 (n=5; biological replicates) and H3K27me3 (n=9; biological replicates) at the TSS on Asns genomic locus in control and EPZ treated condition of in vitro-derived NPCs. One-sided paired Wilcoxon statistical test was performed, error bars represent SEM. *p<0.04.
Figure 7C) Ex utero electroporated cells expressing GFP (green) or TUBB3 (magenta) in control (Con; pCMV-GFP) or in ASNS overexpression (ASNS OE; pCMV-ASNS-GFP) condition 24 h after slice culture. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily a GFP and TUBB3 double positive cell upon ASNS overexpression. Dotted shapes indicate the border of cell soma based on GFP expression. D) Fraction of TUBB3-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from independent experiments (n=3). Student's t-test, **p<0.01, error bars represent SEM.E) Representative images of mCherry (red) or TBR1-expressing (green) cells in control (Con; pCAG-mCherry) or in ASNS overexpression (ASNS OE; pCAG-ASNS) condition, 48 h after in utero electroporation. Scale bars: 20 µm. Dotted lines mark ventricular surface of the tissue. Magnifications on the right show exemplarily mCherry and TBR1 double or single positive cells upon ASNS overexpression. F) Fraction of TBR1-positive and -negative cells expressed as percentage of total cells in Con and upon ASNS OE. Quantifications were made from 1141 cells in control group and 606 cells in ASNS OE group. Fisher's exact test, ****p<0.0001.
Figure 1Live cell microscopy of U-2 OS-TetO cells expressing rTetR-GFP (ctrl), or rTetR-GFP-Kin14VIb (Kin14VIb) (B) Representative stills showing the most frequently observed metaphase localization and daughter nuclei distribution of the duplicated TetO locus (white arrowheads). Chromatin was visualized through expression of H2B-mCherry, GFP depicts the TetO locus. Due to the presence of a predicted NES sequence in Kin14VIb, the TetO locus only becomes visible at NEB in Kin14VIb-expressing cells. Time = h:min, scale bar = 5 µm. Schemes illustrating the metaphase localization of the TetO locus and its subsequent distribution over daughter nuclei are shown on the right.
Figure 2(C) Quantification of the fraction of ctrl or Kin14VIb-expressing cells with the TetO locus observed outside the metaphase plate (TetO out). (D) Deconstruction of (C). Fraction of cells with the indicated orientation of the duplicated TetO locus and TetO chromosome. Data information: Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. (C, D): N ≥ 29 cells per condition, per experiment, ****P<0.0001; Fisher's exact test.E. Quantification of the number of cells with Mad1-positive KTs nearby the TetO locus. In case the KTs of the TetO chromosome were aligned, we scored whether KTs in the vicinity of the TetO locus were Mad1+. In case the KTs of the TetO chromosome could be distinguished in rTetR-GFP-Kin14VIb-expressing cells (with either orthogonal or parallel oriented KTs), we scored if at least one of the KTs was Mad1+. Data information: Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. (E): N ≥27 cells per condition, per experiment, (ns) not significant; Fisher's exact test.
Figure 3A-D (A, C) Frequencies of the different types of segregation errors observed during anaphase (A) and late anaphase/telophase (C). Schemes illustrating the observed types of anaphase errors are shown on the right of (A). Cells within the main TetO locus distribution categories in control (1-1), and Kin14VIb (2-0) conditions were scored (see also Fig EV1A). The majority of Kin14VIb-expressing anaphases display at least one stretched arm (CENP-C in line with TetO locus at the pole) or a bridge in line with the TetO locus at the pole ("stretched arm or bridge in line"), some of them with additional bridges (+). (B, D) IF images for CENP-C, GFP, H3S10ph of U-2 OS TetO cells in anaphase (B) and late anaphase or telophase (D), expressing either rTetR-GFP or rTetR-GFP-Kin14VIb. Images (maximum intensity projections) represent the most frequently observed anaphase and telophase errors in cells with either a 1-1 (majority of events in ctrl), or 2-0 (majority of events in Kin14VIb) TetO locus distribution. Scale bar = 5 µm. Yellow arrowheads indicate TetO locus, red arrowheads CENP-C. Data information: (A, C): Means ± S.E.M. of 3 biological replicates are shown. Dots represent the values of each experiment. N= 41-89 cells per condition, per experiment ****P<0.0001, (ns) not significant; Fisher's exact test. (B, D): for optimal visibility of the TetO foci, the brightness and contrast of all channels were linearly adjusted for individual cells.
Figure 4A. Representative images of U-2 OS TetO anaphases/telophases expressing rTetR-GFP or rTetR-GFP-Kin14VIb with FISH-marked centromeres of chromosome 1 (chr1-CEN). White and yellow arrowheads: chr1-CEN segregated into the main masses of segregating chromosomes; red arrowheads: chr1-CEN FISH signal fragmented or splitted in a bridge (bridging); chr1-CEN lagging between the two main masses of segregating chromosomes (lagging); chr1-CEN late arrival (late-equal: one CEN focus in but near the edge of a separated chromosome mass and connected to a bridge). For clarity, the maximum intensity projection of a subset of z stacks (6-12/64) is shown. Scale bar = 5 µm. B. Frequency of the different types of errors observed in A. The mean (bars) and individual values (dots) of 2 independent experiments are shown. Data information: In (A), for optimal visibility of the chr1-CEN FISH foci, the brightness and contrast were linearly adjusted for individual cells. (B): n ≥ 45 cells per condition, per experiment. Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. * P<0.05, (ns) not significant. Fisher's exact test.