Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 1. Effect of caffeine on cell survival and the cell cycle. (A) Influence of various concentrations of caffeine on igm482 growth. For each data point, two measurements of cell density were taken from each of three flasks. The mean cell density ± standard error of the mean is presented. If the standard error bar is not indicated, it is contained within the marker. (B) DNA content of igm482 cells, assessed by fluorescence-activated cell sorting (FACS) following propidium iodide staining of fixed cells. An example of untreated and treated DNA histograms is presented along with graphical analysis of a minimum of 3 replicates, each with a minimum of 19,000 cells analyzed. igm482 cells were treated with 5 mM caffeine for 24 h where indicated. Percentage of cells in each cell cycle phase was determined by gate analysis using Cellquest Pro software. The mean percentage of cells in each cell cycle phase ± standard error of the mean is presented. Percentage of cells in G2 are contained within brackets to indicate that they are a subset of S/G2. Differences between untreated and treated cells were determined for each cell cycle phase by t-tests and significance is indicated by asterisk (p ≤0.05). (C) igm482 cells were treated with 5 mM caffeine for 24 h. An assessment of apoptosis was performed by measuring the liberation of fluorescent AMC from Ac-DEVD-AMC, a fluorogenic substrate specific for caspase-3. Two measurements of DEVDase activity (relative activity/min/μg protein) were recorded for each sample and the assay was repeated three times. DEVDase activity was standardized relative to untreated igm482 cells and the mean ± standard error of the mean is presented. Means indicated by asterisk are significantly different according to one- way ANOVA and t-test (p ≤0.05). Abbreviations: UT, untreated; T, treated.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques: Marker, Fluorescence, FACS, Staining, Software, Activity Assay

Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 2. Caffeine affects the DNA Damage Response (DDR). (A) igm482 cells were treated with 5 mM caffeine for the indicated time-course and whole cell-extracts were analyzed by Western blot for DDR-related proteins. (B) RT-PCR analysis of p53 transcript levels in untreated igm482 cells or in igm482 cells treated with 5 mM caffeine for the indicated time-course. Densitometric ana- lysis of band intensity was used to determine p53 transcript levels relative to those of the single copy, chromosomal immunoglobulin μ gene. The p53/μ ratio in the treated cells was standardized re- lative to untreated cells. (D) Proteasome inhibitor analysis in igm482 using 5 mM caffeine and/or MG132 (10μM) for 2 h. Whole-cell extracts were then analyzed for p53 by Western blot. Abbreviations: UT, untreated.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Lysis

Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 3. Caffeine affects levels of HR proteins. (A) igm482 cells were treated with varying amounts of caffeine for the time-course indicated. Whole-cell extracts were analyzed by Western blot for Rad51. (B) HeLa and CHO-WBL cells were treated for 24 h with 5 mM caffeine and whole-cell extracts were analyzed by Western blot for Rad51. (C) igm482 and WT5 cells were exposed to 5 mM caffeine for the indicated time-course. Whole-cell extracts were analyzed by Western blot for Rad51 and Brca2. The membranes used for Brca2 Western blot analyses were stained in India ink and a portion of each is presented as a loading control. (D) RT-PCR analysis of Rad51 and Brca2 transcript levels in untreated igm482 cells or in igm482 cells treated with 5 mM caffeine for the indicated time-course. Densitometric analysis of band intensity was used to determine transcript levels relative to those of the single copy, chromosomal immunoglobulin μ gene. The Rad51/μ and Brca2/μ ratios in the treated cells were standardized relative to untreated cells. (E) igm482, p53 kd#3 and p53 kd#6 were exposed to 5 mM caffeine for 24 h. Whole cell extracts were analyzed by Western blot for p53, phosphor-Ser18-p53 and Rad51. Densitometric analysis of band intensity was first used to standardize p53 and Rad51 to ß-actin. Rad51/p53 ratios were then determined for each cell line. Unadjusted ratios are presented along with adjusted ratios where p53 kd#3 and p53kd#6 untreated and treated ratios are expressed relative to igm482 untreated and treated ratios respectively. Abbreviations: UT, untreated; T, treated.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques: Western Blot, Staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 4. Early steps of HR are stimulated by caffeine. (A) Effect of caffeine on new DNA synthesis (3′ polymerization) in igm482 cells exposed to 5 mM caffeine for 2 h, electroporated and then returned to 5 mM caffeine for the duration of the experiment. Each data point represents the mean fold change in 3′ polymerization/vector backbone ± standard error of the mean of treated cells compared to untreated cells. At least three independent electroporations with replicate PCR reactions were analyzed. Differences in mean fold-change were determined at each time point by t-tests and significance is indicated by asterisk (p ≤0.05). (B) Graphical re- presentation of DNA content of igm482 cells, assessed by FACS following propidium iodide staining of fixed cells. The percentage of cells in each cell cycle phase was determined by gate analysis using CellQuest Pro software. For each data point, a minimum of 19,000 cells were analyzed in each of a minimum of three re- plicates. The mean percentage of cells ± standard error of the mean is presented. If the standard error bar is not indicated, it is contained within the marker. Duration of exposure to 5 mM caf- feine and the corresponding 3′polymerization time is indicated. Differences between means at each time point were determined by t-tests and significance is indicated by asterisk (p ≤0.05). Abbreviations: UT, untreated; E, electroporation; ns, non-sig- nificant.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques: DNA Synthesis, Plasmid Preparation, Staining, Software, Marker, Electroporation

Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 5. Brief caffeine treatment elevates the early steps in HR. (A) igm482 cells were treated with 5 mM caffeine for 2 h and then transferred into caffeine-free medium. Whole cell extracts were sampled at the indicated recovery times and analyzed by Western blot for Rad51, p53 and phosphoSer18-p53. (B) Graphical re- presentation of DNA content of igm482 cells following a 2 h caffeine treatment and sub- sequent transfer into caffeine-free media, as- sessed by FACS following propidium iodide staining of fixed cells. Percentage of cells in each cell cycle phase was determined by gate analysis using CellQuest Pro software. A minimum of 19,000 cells were analyzed in a minimum of 3 replicates for each data point. The mean percentage of cells ± standard error of the mean is presented. If the standard error bar is not indicated, it is contained within the marker. The elapsed recovery time following the 2 h caffeine treatment and the corre- sponding 3′ polymerization time is indicated. Differences between means at each time point were determined by t-tests and significance is indicated by asterisk (p ≤0.05). (C) Effect of a 2 h caffeine treatment on new DNA synthesis (3′ polymerization) in igm482. Each data point represents the mean fold-change of 3′ poly- merization/vector backbone ± standard error of the mean, of treated cells compared to un- treated cells. At least three independent elec- troporations with replicate PCRs were ana- lyzed. Differences in mean fold-change were determined at each time point by t-tests and significance is indicated by asterisk (p ≤0.05). Abbreviations: UT, untreated; E, electropora- tion; ns, non-significant.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques: Western Blot, Staining, Software, Marker, DNA Synthesis, Plasmid Preparation

Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 7. Effects of caffeine on chromatin accessibility. (A) Sensitivity to DNase I digestion in cells that were untreated or treated with caffeine (5 mM) or trichostatin A (300 μM). Saturated pixels have been highlighted and indicate areas of undigested genomic DNA. (B) Representative PCR amplification of the μ locus following treatment with DNase I in cells that were untreated, or treated with caffeine (5 mM), trichostatin A (330 μM) or anacardic acid (80 μM). Untreated igm482 cells that were not treated with DNase I are included for reference. (C) The fold change in PCR amplification of the μ locus in igm482 cells following DNase I digestion of cells that were treated with caffeine, trichostatin A or anacardic acid, relative to untreated cells. Each data point represents the mean fold change in PCR amplifica- tion ± standard error of the mean from four independent experiments. Statistical analysis was performed by one-way ANOVA and Tukey’s HSD. Means indicated by the same lower-case letter are not significantly different at p ≤0.05. (D) The fold change in new DNA synthesis (3′ polymerization) in igm482 cells exposed to caffeine, trichostatin A and anacardic acid, relative to untreated cells. Each data point represents the mean fold change in 3′ polymerization/vector backbone ± standard error of the mean at 6 h post-electroporation. Three independent electroporations with replicate PCR reactions were analyzed. Statistical analysis was performed by one-way ANOVA and Tukey’s HSD. Means indicated by the same lower-case letter are not significantly different at p ≤0.05. Abbreviations: UT, untreated.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques: Amplification, DNA Synthesis, Plasmid Preparation, Electroporation

Journal: DNA repair

Article Title: The dichotomous effects of caffeine on homologous recombination in mammalian cells.

doi: 10.1016/j.dnarep.2020.102805

Figure Lengend Snippet: Fig. 8. Effects of caffeine on illegitimate recombination. The fre- quency of illegitimate recombination in untreated igm482 cells and those treated with 5 mM caffeine for 2 h or for 24 h is pre- sented. Each data point represents the mean frequency of illegi- timate recombination ± standard error of the mean from seven independent experiments. Differences in illegitimate recombina- tion were determined by the GLIMMIX procedure, log link and Tukey’s- HSD. Means indicated by the same lower-case letter are not significantly different at p ≤0.05. Abbreviations: UT, un- treated; T, treated.

Article Snippet: However, in repeated experiments, we found no evidence for this Rad51 cleavage product in caffeine-treated igm482 cells using two different anti-Rad51 antibodies (14B4, Abcam; H-92, Santa Cruz Biotechnology) (data not shown).

Techniques:

Journal: SpringerPlus

Article Title: Assessing developmental toxicity of caffeine and sweeteners in medaka ( Oryzias latipes )

doi: 10.1186/s40064-015-1284-0

Figure Lengend Snippet: Abbreviations and concentrations of chemicals used in this study

Article Snippet: Sucrose (PA34230, purity > 99 %) was purchased from Panreac (Barcelona, Spain); ASP (228650050, purity > 98 %), SAC (149001000, purity > 98 %), and CAF (108160100, purity > 98.5 %) were purchased from Acros Organics (Geel, Belgium).

Techniques: Molecular Weight, Concentration Assay, Control

Journal: SpringerPlus

Article Title: Assessing developmental toxicity of caffeine and sweeteners in medaka ( Oryzias latipes )

doi: 10.1186/s40064-015-1284-0

Figure Lengend Snippet: Effects of sucrose (SUC), aspartame (ASP), saccharin (SAC), caffeine (CAF), and CAF combined with each sweetener on medaka development at 3 days post fertilization or at hatch. Data were percents of control values and expressed as mean ± SEM. *Significantly different from the control or between pairs of groups, p < 0.05; **p < 0.01; ***p < 0.001. The abbreviations and concentrations of the substances are listed in Table

Article Snippet: Sucrose (PA34230, purity > 99 %) was purchased from Panreac (Barcelona, Spain); ASP (228650050, purity > 98 %), SAC (149001000, purity > 98 %), and CAF (108160100, purity > 98.5 %) were purchased from Acros Organics (Geel, Belgium).

Techniques: Control

Journal: SpringerPlus

Article Title: Assessing developmental toxicity of caffeine and sweeteners in medaka ( Oryzias latipes )

doi: 10.1186/s40064-015-1284-0

Figure Lengend Snippet: Summary of biological responses in medaka embryos exposed to various substances during development

Article Snippet: Sucrose (PA34230, purity > 99 %) was purchased from Panreac (Barcelona, Spain); ASP (228650050, purity > 98 %), SAC (149001000, purity > 98 %), and CAF (108160100, purity > 98.5 %) were purchased from Acros Organics (Geel, Belgium).

Techniques: