chlorodeoxyuridine cldu  (Valiant Co Ltd)

Journal: The Journal of Cell Biology

Article Title: DNA combing versus DNA spreading and the separation of sister chromatids

doi: 10.1083/jcb.202305082

Figure Lengend Snippet: Schematic and model for defining the outcome of sister chromatid alignment versus separation. (A) Use of halogenated thymidine analogs to distinguish between sister chromatids during S phase. RPE-1 and U2OS cells are incubated with 1 µM CldU for 24 h (first round of replication), chased with fresh media for 1 h, and incubated with 200 µM IdU for 30 min (second round of replication). (B) Predicted outcomes for sister chromatid alignment (left) versus separation (right). If sister chromatids remain aligned/adjacent to each other (left), they will result in DNA fibers that are red–green–red if there is no breakage, breakage at a, or breakage at b, and in fibers that are red–green upon breakage at both at a and b. In contrast, if the sister chromatids become separated (right), they will result in DNA fibers that are red–green–red if there is no breakage, red–green upon breakage at a, red–green–red and green only upon breakage at b, and red–green and green only upon breakage at both a and b.

Article Snippet: Exponentially growing hTERT-RPE1 (RRID:CVCL_4388) cells were pulse-labeled with 30 μM CldU (5-Chloro-2′-deoxyuridine, 105478; MP Biomedicals) and 250 μM IdU (5-Iodo-2′-deoxyuridine, I7125; Sigma-Aldrich) as described in the figure legends.

Techniques: Incubation

Journal: The Journal of Cell Biology

Article Title: DNA combing versus DNA spreading and the separation of sister chromatids

doi: 10.1083/jcb.202305082

Figure Lengend Snippet: (Related to ): Examples of DNA fiber images collected with the DNA combing, spreading, and hybrid spreading techniques. (A) Images collected with DNA combing. The numbers 1, 2, and 3 denote the examples of red–green–red, red–green, and green-only fibers shown in . (B) Images collected with DNA spreading. The numbers 1, 2, and 3 denote the examples of red–green–red, red–green, and green-only fibers shown in . (C) Images collected with the hybrid spreading technique. The numbers 1, 2, and 3 denote the examples of red–green–red, red–green, and green-only fibers shown in . In all cases, when we score red–green–red or red–green fibers, the red signal is continuous and extends through the green tracts, confirming that our labeling originates from two separate replication cycles. The reason why we do not see yellow tracts when the CldU and IdU overlap is that the IdU signal (green) is stronger than the CldU signal (red) due to the different efficiencies of the two antibodies (see also Materials and methods).

Article Snippet: Exponentially growing hTERT-RPE1 (RRID:CVCL_4388) cells were pulse-labeled with 30 μM CldU (5-Chloro-2′-deoxyuridine, 105478; MP Biomedicals) and 250 μM IdU (5-Iodo-2′-deoxyuridine, I7125; Sigma-Aldrich) as described in the figure legends.

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: DNA combing versus DNA spreading and the separation of sister chromatids

doi: 10.1083/jcb.202305082

Figure Lengend Snippet: DNA combing, but not DNA spreading, can detect DNA single-strand gaps within individual sister chromatids. (A) Schematic depicting the use of pulse-labeling and FEN1 inhibitor (FEN1i) to detect single-strand gaps at unligated Okazaki fragments. (B) Dot plots of IdU/CldU ratios in cells treated ± FEN1i and ± S1 nuclease as indicated. Number of biological repeats N = 2. Bars represent the median values. At least 100 tracts were scored for each sample. Statistics: Kruskal–Wallis test. ns, non-significant, *P ≤ 0.05, ****P ≤ 0.0001.

Article Snippet: Exponentially growing hTERT-RPE1 (RRID:CVCL_4388) cells were pulse-labeled with 30 μM CldU (5-Chloro-2′-deoxyuridine, 105478; MP Biomedicals) and 250 μM IdU (5-Iodo-2′-deoxyuridine, I7125; Sigma-Aldrich) as described in the figure legends.

Techniques: Labeling

Journal: iScience

Article Title: Ethanol induces replication fork stalling and membrane stress in immortalized laryngeal cells

doi: 10.1016/j.isci.2023.108564

Figure Lengend Snippet: HuLa-PC cells show a G1 phase delay and a slower replication fork progression when exposed to ethanol (A) Heatmap with expression changes of genes involved in G1 to S cell cycle phase transition in 250 mM ethanol-treated HuLa-PC cells. Expression changes are represented as log2 fold changes relative to the control cells, treated with 0 mM ethanol. Hierarchical clustering was performed by average linkage. (B) Effect of ethanol (EtOH) on cell cycle distribution in Hula-PC cells. Two cell cycle profiles obtained after 24 h of ethanol exposure are depicted as an example. The graph indicates the percentage of cells in the G1 cell cycle phase in medium with or without ethanol. At least three individual biological replicates were measured, the bar indicates the average with standard deviation. Significance was assessed by a two-way ANOVA and a Šidák posthoc test to the respective control at the same time point. ∗∗∗ p-values <0.001, ∗∗∗∗p < 0.0001. (C) Effect of ethanol (EtOH) on population doubling time in HuLa-PC cells. Four independent biological replicates were measured, the bar indicates the average with standard deviation. At 400 mM ethanol, no growth was observed which is indicated by a capped bar. Statistical significance was determined by a one-way ANOVA with a Dunnett posthoc test. ∗∗∗∗p < 0.0001. (D) DNA labeling scheme for the DNA fiber analysis. Cells were consecutively treated with 5-iodo-2′-deoxyuridine (IdU) and 5-chloro-2′-deoxyuridine (CldU). Both pulses were exactly 30 min and ethanol (EtOH) or MMS was added during the second pulse only. (E) Effect of ethanol (EtOH) or MMS on replication fork progression in HuLa-PC cells assessed by a DNA fiber analysis. Cells were labeled and treated as specified in panel (D). The ratio of IdU/CldU track length is depicted. For each condition, four independent biological replicates were assessed, with a minimum of 150 fibers each. Each dot corresponds to a measurement of an individual DNA fiber, the black bar represents the average IdU/CldU ratio. The fold change with respect to the control is also indicated on the graph. Number of scored fibers: Control = 990; MMS = 520; EtOH 100 mM = 549; EtOH 250 mM = 607; EtOH 400 mM = 616. Statistical significance was assessed by a Welch-corrected one-way ANOVA with Games-Howell posthoc test on log-transformed ratios. ∗∗∗∗p < 0.0001.

Article Snippet: 5-chloro-2′-deoxyuridine , MP Biomedicals , Cat# HY-112669.

Techniques: Expressing, Sublimation, Standard Deviation, DNA Labeling, Labeling, Transformation Assay