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frap data analysis  (Nikon)


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    Nikon frap data analysis
    Frap Data Analysis, supplied by Nikon, used in various techniques. Bioz Stars score: 99/100, based on 40098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/frap+data+analysis/NIS-Elements/pm39824183-1012-0-5
    Average 99 stars, based on 40098 article reviews
    frap data analysis - by Bioz Stars, 2026-10
    99/100 stars

    Images

    Related Articles

    Confocal:

    Article Title: Mechanisms of Hexavalent Chromium-Induced Lung Carcinogenesis: DNA Double-Strand Breaks and DNA Repair Inhibition in a Guinea Pig Model.
    Article Snippet: .. These lung regions were imaged using a Nikon A1 Confocal Microscope Ti with the NIS Elements software (ver. ..

    Article Title: Sirt3 deficiency impairs AMPK-Acc1 antilipogenic signaling in female adipocytes.
    Article Snippet: 1 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 2 Division of Materials Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia 3 Faculty of Science, Department of Biology, University of Zagreb, Zagreb, Croatia 4 Faculty of Biology, Center for Electron Microscopy, University of Belgrade, Belgrade, Serbia Abstract Background Background: Biological sex influences adipose tissue function, while impaired adipogenesis contributes to adipose dysfunction.. Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase involved in oxidative metabolism and stress adaptation, remains insufficiently defined in adipogenic differentiation.. We investigated how Sirt3 and sex shape adipogenic phenotype in mouse embryonic fibroblast (MEF)-derived adipocytes.

    Article Title: Trichoderma harzianum M2 Induces Systemic Resistance to Fusarium Crown Rot and Remodels Root Architecture to Promote Phosphorus Uptake in Wheat.
    Article Snippet: .. Putative positive transformants were further verified by PCR amplification of the GFP gene, and GFP fluorescence signal was detected using a Nikon confocal microscope (AX NIS-Elements 5.4, Tokyo, Japan) to confirm GFP expression. ..

    Microscopy:

    Article Title: Mechanisms of Hexavalent Chromium-Induced Lung Carcinogenesis: DNA Double-Strand Breaks and DNA Repair Inhibition in a Guinea Pig Model.
    Article Snippet: .. These lung regions were imaged using a Nikon A1 Confocal Microscope Ti with the NIS Elements software (ver. ..

    Article Title: Peripheral nerve injury alters metabolic function and neuronal excitability through the Lin28/let-7 pathway
    Article Snippet: .. Image pairs were continually acquired at 340-nm 100-ms excitation and 380-nm 200-ms excitation at 1.5-s intervals using an inverted fluorescence microscope (TE200, Nikon) equipped with a complementary metal-oxide semiconductor camera (Neo, Andor) and an automated filter wheel (Lambda), controlled by the NIS elements software (Nikon), and emission at 340/380 nm was calculated. .. Cells were imaged during continuous perfusion with CIB and then challenged with sequential 30-s applications of 15 mM KCl, capsaicin (10 nM; Sigma-Aldrich), and 50 mM KCl, with 3-min intervening washouts with CIB.

    Article Title: Sirt3 deficiency impairs AMPK-Acc1 antilipogenic signaling in female adipocytes.
    Article Snippet: 1 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 2 Division of Materials Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia 3 Faculty of Science, Department of Biology, University of Zagreb, Zagreb, Croatia 4 Faculty of Biology, Center for Electron Microscopy, University of Belgrade, Belgrade, Serbia Abstract Background Background: Biological sex influences adipose tissue function, while impaired adipogenesis contributes to adipose dysfunction.. Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase involved in oxidative metabolism and stress adaptation, remains insufficiently defined in adipogenic differentiation.. We investigated how Sirt3 and sex shape adipogenic phenotype in mouse embryonic fibroblast (MEF)-derived adipocytes.

    Article Title: Prenatal microplastic exposure-induced testicular developmental toxicity and its long-term hazards in male rat offspring.
    Article Snippet: The pervasive environmental presence of microplastics (MPs) raises concerns regarding their potential impacts on reproductive health, particularly in the context of prenatal exposure.. This study aimed to establish a polystyrene microplastics (PS-MPs) exposure model in pregnant rats and to investigate the subsequent effects on the growth, hormonal profiles, steroidogenic enzyme expression, and testicular histology of male offspring from birth to adulthood.. Pregnant rats were administered PS-MPs at doses of 1 mg/kg/day (MPs-L) and 10 mg/kg/day (MPs-H) during gestational days 10–20.

    Article Title: Discovery of a Virulent Population of Meloidogyne graminicola Overcoming the Rice Resistance Gene MG1 and Population Genetic Analysis Based on mtCOI Sequences.
    Article Snippet: .. Measurements and photographs were https://doi.org/10.3390/plants15193031 taken with a Nikon ECLIPSE Ni microscope equipped with a Nikon Digital Sight Camera and NIS-Elements BR 4.13.05 software (Nikon, Tokyo, Japan). ..

    Article Title: Trichoderma harzianum M2 Induces Systemic Resistance to Fusarium Crown Rot and Remodels Root Architecture to Promote Phosphorus Uptake in Wheat.
    Article Snippet: .. Putative positive transformants were further verified by PCR amplification of the GFP gene, and GFP fluorescence signal was detected using a Nikon confocal microscope (AX NIS-Elements 5.4, Tokyo, Japan) to confirm GFP expression. ..

    Software:

    Article Title: Mechanisms of Hexavalent Chromium-Induced Lung Carcinogenesis: DNA Double-Strand Breaks and DNA Repair Inhibition in a Guinea Pig Model.
    Article Snippet: .. These lung regions were imaged using a Nikon A1 Confocal Microscope Ti with the NIS Elements software (ver. ..

    Article Title: Peripheral nerve injury alters metabolic function and neuronal excitability through the Lin28/let-7 pathway
    Article Snippet: .. Image pairs were continually acquired at 340-nm 100-ms excitation and 380-nm 200-ms excitation at 1.5-s intervals using an inverted fluorescence microscope (TE200, Nikon) equipped with a complementary metal-oxide semiconductor camera (Neo, Andor) and an automated filter wheel (Lambda), controlled by the NIS elements software (Nikon), and emission at 340/380 nm was calculated. .. Cells were imaged during continuous perfusion with CIB and then challenged with sequential 30-s applications of 15 mM KCl, capsaicin (10 nM; Sigma-Aldrich), and 50 mM KCl, with 3-min intervening washouts with CIB.

    Article Title: Sirt3 deficiency impairs AMPK-Acc1 antilipogenic signaling in female adipocytes.
    Article Snippet: 1 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 2 Division of Materials Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia 3 Faculty of Science, Department of Biology, University of Zagreb, Zagreb, Croatia 4 Faculty of Biology, Center for Electron Microscopy, University of Belgrade, Belgrade, Serbia Abstract Background Background: Biological sex influences adipose tissue function, while impaired adipogenesis contributes to adipose dysfunction.. Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase involved in oxidative metabolism and stress adaptation, remains insufficiently defined in adipogenic differentiation.. We investigated how Sirt3 and sex shape adipogenic phenotype in mouse embryonic fibroblast (MEF)-derived adipocytes.

    Article Title: Prenatal microplastic exposure-induced testicular developmental toxicity and its long-term hazards in male rat offspring.
    Article Snippet: The pervasive environmental presence of microplastics (MPs) raises concerns regarding their potential impacts on reproductive health, particularly in the context of prenatal exposure.. This study aimed to establish a polystyrene microplastics (PS-MPs) exposure model in pregnant rats and to investigate the subsequent effects on the growth, hormonal profiles, steroidogenic enzyme expression, and testicular histology of male offspring from birth to adulthood.. Pregnant rats were administered PS-MPs at doses of 1 mg/kg/day (MPs-L) and 10 mg/kg/day (MPs-H) during gestational days 10–20.

    Article Title: Discovery of a Virulent Population of Meloidogyne graminicola Overcoming the Rice Resistance Gene MG1 and Population Genetic Analysis Based on mtCOI Sequences.
    Article Snippet: .. Measurements and photographs were https://doi.org/10.3390/plants15193031 taken with a Nikon ECLIPSE Ni microscope equipped with a Nikon Digital Sight Camera and NIS-Elements BR 4.13.05 software (Nikon, Tokyo, Japan). ..

    Article Title: Expanded characterization of the valproic acid zebrafish larval model of autism spectrum disorder: Insights into dysregulation in the gut
    Article Snippet: Magnesium sulfate (208094, Sigma‐Aldrich, Wisconsin, United States; 33.2 mmol/L) and atropine (A0257, Sigma‐Aldrich, St Louis, United States; 5 mmol/L) were included as positive controls to mimic accelerated and delayed gut motility, respectively. .. Larvae were anesthetized and orientated as described in the NR protocol before live images of gut fluorescence were acquired at 100× magnification (Nikon ECLIPSE Ti2 inverted microscope, NiS‐Elements D, version 5.30.02 software). .. Brightfield images of the gut were overlaid with fluorescent images to accurately demarcate the region of interest before quantification of gut fluorescent intensity (using ImageJ software).

    Fluorescence:

    Article Title: Peripheral nerve injury alters metabolic function and neuronal excitability through the Lin28/let-7 pathway
    Article Snippet: .. Image pairs were continually acquired at 340-nm 100-ms excitation and 380-nm 200-ms excitation at 1.5-s intervals using an inverted fluorescence microscope (TE200, Nikon) equipped with a complementary metal-oxide semiconductor camera (Neo, Andor) and an automated filter wheel (Lambda), controlled by the NIS elements software (Nikon), and emission at 340/380 nm was calculated. .. Cells were imaged during continuous perfusion with CIB and then challenged with sequential 30-s applications of 15 mM KCl, capsaicin (10 nM; Sigma-Aldrich), and 50 mM KCl, with 3-min intervening washouts with CIB.

    Article Title: Prenatal microplastic exposure-induced testicular developmental toxicity and its long-term hazards in male rat offspring.
    Article Snippet: The pervasive environmental presence of microplastics (MPs) raises concerns regarding their potential impacts on reproductive health, particularly in the context of prenatal exposure.. This study aimed to establish a polystyrene microplastics (PS-MPs) exposure model in pregnant rats and to investigate the subsequent effects on the growth, hormonal profiles, steroidogenic enzyme expression, and testicular histology of male offspring from birth to adulthood.. Pregnant rats were administered PS-MPs at doses of 1 mg/kg/day (MPs-L) and 10 mg/kg/day (MPs-H) during gestational days 10–20.

    Article Title: Expanded characterization of the valproic acid zebrafish larval model of autism spectrum disorder: Insights into dysregulation in the gut
    Article Snippet: Magnesium sulfate (208094, Sigma‐Aldrich, Wisconsin, United States; 33.2 mmol/L) and atropine (A0257, Sigma‐Aldrich, St Louis, United States; 5 mmol/L) were included as positive controls to mimic accelerated and delayed gut motility, respectively. .. Larvae were anesthetized and orientated as described in the NR protocol before live images of gut fluorescence were acquired at 100× magnification (Nikon ECLIPSE Ti2 inverted microscope, NiS‐Elements D, version 5.30.02 software). .. Brightfield images of the gut were overlaid with fluorescent images to accurately demarcate the region of interest before quantification of gut fluorescent intensity (using ImageJ software).

    Article Title: Trichoderma harzianum M2 Induces Systemic Resistance to Fusarium Crown Rot and Remodels Root Architecture to Promote Phosphorus Uptake in Wheat.
    Article Snippet: .. Putative positive transformants were further verified by PCR amplification of the GFP gene, and GFP fluorescence signal was detected using a Nikon confocal microscope (AX NIS-Elements 5.4, Tokyo, Japan) to confirm GFP expression. ..

    Automated:

    Article Title: Peripheral nerve injury alters metabolic function and neuronal excitability through the Lin28/let-7 pathway
    Article Snippet: .. Image pairs were continually acquired at 340-nm 100-ms excitation and 380-nm 200-ms excitation at 1.5-s intervals using an inverted fluorescence microscope (TE200, Nikon) equipped with a complementary metal-oxide semiconductor camera (Neo, Andor) and an automated filter wheel (Lambda), controlled by the NIS elements software (Nikon), and emission at 340/380 nm was calculated. .. Cells were imaged during continuous perfusion with CIB and then challenged with sequential 30-s applications of 15 mM KCl, capsaicin (10 nM; Sigma-Aldrich), and 50 mM KCl, with 3-min intervening washouts with CIB.

    Imaging:

    Article Title: Sirt3 deficiency impairs AMPK-Acc1 antilipogenic signaling in female adipocytes.
    Article Snippet: 1 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 2 Division of Materials Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia 3 Faculty of Science, Department of Biology, University of Zagreb, Zagreb, Croatia 4 Faculty of Biology, Center for Electron Microscopy, University of Belgrade, Belgrade, Serbia Abstract Background Background: Biological sex influences adipose tissue function, while impaired adipogenesis contributes to adipose dysfunction.. Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase involved in oxidative metabolism and stress adaptation, remains insufficiently defined in adipogenic differentiation.. We investigated how Sirt3 and sex shape adipogenic phenotype in mouse embryonic fibroblast (MEF)-derived adipocytes.

    Inverted Microscopy:

    Article Title: Sirt3 deficiency impairs AMPK-Acc1 antilipogenic signaling in female adipocytes.
    Article Snippet: 1 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 2 Division of Materials Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia 3 Faculty of Science, Department of Biology, University of Zagreb, Zagreb, Croatia 4 Faculty of Biology, Center for Electron Microscopy, University of Belgrade, Belgrade, Serbia Abstract Background Background: Biological sex influences adipose tissue function, while impaired adipogenesis contributes to adipose dysfunction.. Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase involved in oxidative metabolism and stress adaptation, remains insufficiently defined in adipogenic differentiation.. We investigated how Sirt3 and sex shape adipogenic phenotype in mouse embryonic fibroblast (MEF)-derived adipocytes.

    Article Title: Expanded characterization of the valproic acid zebrafish larval model of autism spectrum disorder: Insights into dysregulation in the gut
    Article Snippet: Magnesium sulfate (208094, Sigma‐Aldrich, Wisconsin, United States; 33.2 mmol/L) and atropine (A0257, Sigma‐Aldrich, St Louis, United States; 5 mmol/L) were included as positive controls to mimic accelerated and delayed gut motility, respectively. .. Larvae were anesthetized and orientated as described in the NR protocol before live images of gut fluorescence were acquired at 100× magnification (Nikon ECLIPSE Ti2 inverted microscope, NiS‐Elements D, version 5.30.02 software). .. Brightfield images of the gut were overlaid with fluorescent images to accurately demarcate the region of interest before quantification of gut fluorescent intensity (using ImageJ software).

    Analysis:

    Article Title: Sirt3 deficiency impairs AMPK-Acc1 antilipogenic signaling in female adipocytes.
    Article Snippet: 1 Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, Croatia 2 Division of Materials Chemistry, Rudjer Boskovic Institute, Zagreb, Croatia 3 Faculty of Science, Department of Biology, University of Zagreb, Zagreb, Croatia 4 Faculty of Biology, Center for Electron Microscopy, University of Belgrade, Belgrade, Serbia Abstract Background Background: Biological sex influences adipose tissue function, while impaired adipogenesis contributes to adipose dysfunction.. Sirtuin 3 (Sirt3), a mitochondrial NAD+-dependent deacetylase involved in oxidative metabolism and stress adaptation, remains insufficiently defined in adipogenic differentiation.. We investigated how Sirt3 and sex shape adipogenic phenotype in mouse embryonic fibroblast (MEF)-derived adipocytes.

    Light Microscopy:

    Article Title: Leptin promotes obesity‑related osteoarthritis progression via CD44/JAK2/STAT3 axis‑mediated chondrocyte apoptosis.
    Article Snippet: .. Following dehydration in increasing ethanol concentrations (70‐100%) and xylene clearing, the slides were mounted with neutral balsam and images were captured under a Nikon Eclipse 80i light microscope (Nikon Corporation,) using NIS‐Elements BR software (version 5.42; Nikon Corporation). ..

    Polymerase Chain Reaction:

    Article Title: Trichoderma harzianum M2 Induces Systemic Resistance to Fusarium Crown Rot and Remodels Root Architecture to Promote Phosphorus Uptake in Wheat.
    Article Snippet: .. Putative positive transformants were further verified by PCR amplification of the GFP gene, and GFP fluorescence signal was detected using a Nikon confocal microscope (AX NIS-Elements 5.4, Tokyo, Japan) to confirm GFP expression. ..

    Amplification:

    Article Title: Trichoderma harzianum M2 Induces Systemic Resistance to Fusarium Crown Rot and Remodels Root Architecture to Promote Phosphorus Uptake in Wheat.
    Article Snippet: .. Putative positive transformants were further verified by PCR amplification of the GFP gene, and GFP fluorescence signal was detected using a Nikon confocal microscope (AX NIS-Elements 5.4, Tokyo, Japan) to confirm GFP expression. ..

    Expressing:

    Article Title: Trichoderma harzianum M2 Induces Systemic Resistance to Fusarium Crown Rot and Remodels Root Architecture to Promote Phosphorus Uptake in Wheat.
    Article Snippet: .. Putative positive transformants were further verified by PCR amplification of the GFP gene, and GFP fluorescence signal was detected using a Nikon confocal microscope (AX NIS-Elements 5.4, Tokyo, Japan) to confirm GFP expression. ..



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    Live-cell single-molecule microscopy reveals fraction of stably bound PARP1 and PARP2 in undamaged cells (A) (i) Schematic describing the covalent binding of JF646 dye to the HaloTag. (ii) Sample cropped frames from a representative 97 Hz SPT movie depicting the trajectory of a single PARP1 molecule. The 640 nm excitation laser was used continuously for imaging while the camera exposure time was 10.3 ms. (B and C) Single-particle trajectories (length of >2) over 30 s for Halo-PARP1 (in B) or Halo-PARP2 (in C) in a single representative nucleus. (D) Fraction bound ( F bound ) of Halo-PARP1 and Halo-PARP2 in undamaged cells inferred from Spot-On’s three-state model fitting to 97 Hz SPT data. Bar graphs show the mean F bound ± SEM obtained from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates (represented by dots), each of which were fitted separately. Statistical difference between the two groups was determined using unpaired t-test. (E) Cumulative distribution function (CDF) of displacements for Halo-PARP1 and Halo-PARP2 (representative Δ τ = 30 ms) in undamaged cells. Individual curves depict data merged from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates. (F) A log-log plot showing the uncorrected survival probability (1-CDF) of individual Halo-PARP1 and Halo-PARP2 molecules and their respective two-phase exponential model fits (solid curves) to 2 Hz SPT data in undamaged cells. Each curve represents data merged from ≥870 trajectories from ≥13 cells from ≥3 independent replicates. Data acquired for H2B-Halo (11,737 trajectories from ≥40 cells from 10 independent replicates) was used for photobleaching correction and thereby deriving values for τ transient and τ stable (See <xref ref-type=Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from FRAP experiments performed on Halo-PARP1 (blue circles) and Halo-PARP2 (red circles). H2B-Halo (green circles) was used for photobleaching correction. A two-phase exponential model (solid line) was fit to the FRAP data. Error bars represent SD from 11 to 18 cells from ≥3 independent replicates. " width="250" height="auto" />
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    Live-cell single-molecule microscopy reveals fraction of stably bound PARP1 and PARP2 in undamaged cells (A) (i) Schematic describing the covalent binding of JF646 dye to the HaloTag. (ii) Sample cropped frames from a representative 97 Hz SPT movie depicting the trajectory of a single PARP1 molecule. The 640 nm excitation laser was used continuously for imaging while the camera exposure time was 10.3 ms. (B and C) Single-particle trajectories (length of >2) over 30 s for Halo-PARP1 (in B) or Halo-PARP2 (in C) in a single representative nucleus. (D) Fraction bound ( F bound ) of Halo-PARP1 and Halo-PARP2 in undamaged cells inferred from Spot-On’s three-state model fitting to 97 Hz SPT data. Bar graphs show the mean F bound ± SEM obtained from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates (represented by dots), each of which were fitted separately. Statistical difference between the two groups was determined using unpaired t-test. (E) Cumulative distribution function (CDF) of displacements for Halo-PARP1 and Halo-PARP2 (representative Δ τ = 30 ms) in undamaged cells. Individual curves depict data merged from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates. (F) A log-log plot showing the uncorrected survival probability (1-CDF) of individual Halo-PARP1 and Halo-PARP2 molecules and their respective two-phase exponential model fits (solid curves) to 2 Hz SPT data in undamaged cells. Each curve represents data merged from ≥870 trajectories from ≥13 cells from ≥3 independent replicates. Data acquired for H2B-Halo (11,737 trajectories from ≥40 cells from 10 independent replicates) was used for photobleaching correction and thereby deriving values for τ transient and τ stable (See <xref ref-type=Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from FRAP experiments performed on Halo-PARP1 (blue circles) and Halo-PARP2 (red circles). H2B-Halo (green circles) was used for photobleaching correction. A two-phase exponential model (solid line) was fit to the FRAP data. Error bars represent SD from 11 to 18 cells from ≥3 independent replicates. " width="250" height="auto" />
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    APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by <t>FRAP</t> in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad <t>Prism</t> <t>software</t> as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).
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    APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by <t>FRAP</t> in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad <t>Prism</t> <t>software</t> as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).
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    APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by <t>FRAP</t> in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad <t>Prism</t> <t>software</t> as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).
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    Image Search Results


    Live-cell single-molecule microscopy reveals fraction of stably bound PARP1 and PARP2 in undamaged cells (A) (i) Schematic describing the covalent binding of JF646 dye to the HaloTag. (ii) Sample cropped frames from a representative 97 Hz SPT movie depicting the trajectory of a single PARP1 molecule. The 640 nm excitation laser was used continuously for imaging while the camera exposure time was 10.3 ms. (B and C) Single-particle trajectories (length of >2) over 30 s for Halo-PARP1 (in B) or Halo-PARP2 (in C) in a single representative nucleus. (D) Fraction bound ( F bound ) of Halo-PARP1 and Halo-PARP2 in undamaged cells inferred from Spot-On’s three-state model fitting to 97 Hz SPT data. Bar graphs show the mean F bound ± SEM obtained from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates (represented by dots), each of which were fitted separately. Statistical difference between the two groups was determined using unpaired t-test. (E) Cumulative distribution function (CDF) of displacements for Halo-PARP1 and Halo-PARP2 (representative Δ τ = 30 ms) in undamaged cells. Individual curves depict data merged from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates. (F) A log-log plot showing the uncorrected survival probability (1-CDF) of individual Halo-PARP1 and Halo-PARP2 molecules and their respective two-phase exponential model fits (solid curves) to 2 Hz SPT data in undamaged cells. Each curve represents data merged from ≥870 trajectories from ≥13 cells from ≥3 independent replicates. Data acquired for H2B-Halo (11,737 trajectories from ≥40 cells from 10 independent replicates) was used for photobleaching correction and thereby deriving values for τ transient and τ stable (See <xref ref-type=Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from FRAP experiments performed on Halo-PARP1 (blue circles) and Halo-PARP2 (red circles). H2B-Halo (green circles) was used for photobleaching correction. A two-phase exponential model (solid line) was fit to the FRAP data. Error bars represent SD from 11 to 18 cells from ≥3 independent replicates. " width="100%" height="100%">

    Journal: iScience

    Article Title: Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

    doi: 10.1016/j.isci.2022.105779

    Figure Lengend Snippet: Live-cell single-molecule microscopy reveals fraction of stably bound PARP1 and PARP2 in undamaged cells (A) (i) Schematic describing the covalent binding of JF646 dye to the HaloTag. (ii) Sample cropped frames from a representative 97 Hz SPT movie depicting the trajectory of a single PARP1 molecule. The 640 nm excitation laser was used continuously for imaging while the camera exposure time was 10.3 ms. (B and C) Single-particle trajectories (length of >2) over 30 s for Halo-PARP1 (in B) or Halo-PARP2 (in C) in a single representative nucleus. (D) Fraction bound ( F bound ) of Halo-PARP1 and Halo-PARP2 in undamaged cells inferred from Spot-On’s three-state model fitting to 97 Hz SPT data. Bar graphs show the mean F bound ± SEM obtained from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates (represented by dots), each of which were fitted separately. Statistical difference between the two groups was determined using unpaired t-test. (E) Cumulative distribution function (CDF) of displacements for Halo-PARP1 and Halo-PARP2 (representative Δ τ = 30 ms) in undamaged cells. Individual curves depict data merged from ≥42,000 trajectories (>3 detections) from ≥52 cells from ≥5 independent replicates. (F) A log-log plot showing the uncorrected survival probability (1-CDF) of individual Halo-PARP1 and Halo-PARP2 molecules and their respective two-phase exponential model fits (solid curves) to 2 Hz SPT data in undamaged cells. Each curve represents data merged from ≥870 trajectories from ≥13 cells from ≥3 independent replicates. Data acquired for H2B-Halo (11,737 trajectories from ≥40 cells from 10 independent replicates) was used for photobleaching correction and thereby deriving values for τ transient and τ stable (See Table S3 ). (G) Scheme showing 97 Hz and 2 Hz SPT workflow. Three-state model fits to 97 Hz SPT data using Spot-On was used to derive fractions and diffusion coefficients of fast diffusing ( F fast , D fast ), slow diffusing ( F slow , D slow ), and bound PARP ( F bound , D bound ) molecules. Further, 2 Hz SPT data were fit using a two-phase exponential model to derive fractions and duration of transient (Fraction transient, τ transient ) and stable (Fraction stable, τ stable ) PARP binding events. (H and I) Pie chart illustrations summarizing the derivation of overall fractions of Halo-PARP1 (in H) and Halo-PARP2 (in I) engaging in transient and stable binding, slow diffusion, and fast diffusion from 97 to 2 Hz SPT experiments. The bound, slow, and fast-diffusing fractions (in i) were determined using Spot-On’s three-state model fitting to 97 Hz SPT data. The bound fraction (in ii) in Halo-PARP1 and Halo-PARP2 cells was analyzed by 2 Hz SPT and fit to a two-phase exponential model. Data acquired for H2B-Halo were used for photobleaching correction, and a correction factor (see ) was applied to obtain the true fraction of transiently and stably binding Halo-PARP molecules (in iii). These data were compiled together to obtain the overall fractions of endogenous Halo-PARP1 and Halo-PARP2 molecules (in iv). (J) Normalized and photobleaching corrected recovery curves from FRAP experiments performed on Halo-PARP1 (blue circles) and Halo-PARP2 (red circles). H2B-Halo (green circles) was used for photobleaching correction. A two-phase exponential model (solid line) was fit to the FRAP data. Error bars represent SD from 11 to 18 cells from ≥3 independent replicates.

    Article Snippet: FRAP data analysis (MatLab script) , Hansen et al. and Sprague et al. , , https://gitlab.com/anders.sejr.hansen/Mahadevan_2022.

    Techniques: Microscopy, Stable Transfection, Binding Assay, Imaging, Single Particle, Diffusion-based Assay

    Journal: iScience

    Article Title: Dynamics of endogenous PARP1 and PARP2 during DNA damage revealed by live-cell single-molecule imaging

    doi: 10.1016/j.isci.2022.105779

    Figure Lengend Snippet:

    Article Snippet: FRAP data analysis (MatLab script) , Hansen et al. and Sprague et al. , , https://gitlab.com/anders.sejr.hansen/Mahadevan_2022.

    Techniques: Virus, Recombinant, Western Blot, Software, Modification, DNA Extraction

    APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by FRAP in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad Prism software as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).

    Journal: Cancers

    Article Title: Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome

    doi: 10.3390/cancers8050047

    Figure Lengend Snippet: APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by FRAP in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad Prism software as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).

    Article Snippet: The FRAP assay data was analysed in GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) to determine the rate of fluorescence recovery over time, and the relative size of the dynamic and immobile pools of APC ( B–D).

    Techniques: Mutagenesis, Transfection, Fluorescence, Comparison, Software, Knockdown, Western Blot, Control