Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells.
<p>(A) Representative images acquired by a LSM880 Zeiss confocal microscope from Z stack images of mycATRΔC-/- cells (cl8) untreated (NT) or treated with 5 mM of HU for 5 hours. Previously, cells were incubated with 150 µM of IdU for 16 hours for DNA labelling. Cells were fixed with 3% formald...
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| منشور في: |
2025
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| _version_ | 1849927641780453376 |
|---|---|
| author | Gabriel L. A. da Silva (22676649) |
| author2 | Jeziel D. Damasceno (9073663) Jennifer A. Black (14411790) Craig Lapsley (6082646) Richard McCulloch (12918) Luiz R. O. Tosi (9073666) |
| author2_role | author author author author author |
| author_facet | Gabriel L. A. da Silva (22676649) Jeziel D. Damasceno (9073663) Jennifer A. Black (14411790) Craig Lapsley (6082646) Richard McCulloch (12918) Luiz R. O. Tosi (9073666) |
| author_role | author |
| dc.creator.none.fl_str_mv | Gabriel L. A. da Silva (22676649) Jeziel D. Damasceno (9073663) Jennifer A. Black (14411790) Craig Lapsley (6082646) Richard McCulloch (12918) Luiz R. O. Tosi (9073666) |
| dc.date.none.fl_str_mv | 2025-11-24T18:33:13Z |
| dc.identifier.none.fl_str_mv | 10.1371/journal.pgen.1011899.g003 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/figure/Replication_stress_leads_to_ssDNA_accumulation_which_affects_the_mechanisms_influencing_cell_cycle_progression_in_mycATR_C-_-_cells_/30697536 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biophysics Biochemistry Microbiology Cell Biology Genetics Molecular Biology Developmental Biology Science Policy Infectious Diseases Biological Sciences not elsewhere classified Physical Sciences not elsewhere classified major &# 8217 div >< p data reveals loss cells possess mechanisms remarkably plastic genome cas9 genome editing like family acts protein kinase ataxia atr depends upon delayed dna replication stranded dna accumulation leishmania major </ dna damage kinase dna damage genome stability leishmania </ dna metabolism dna injuries atr acts replication stress replication impediments work shows whose integrity terminal knockout smaller chromosomes protozoan parasite phosphatidylinositol 3 nuclear localisation mycatrδc -/-). mycatr ), master regulator larger chromosomes key role increased levels functional relevance eukaryotic response deletion results constantly challenged atr plays atr homolog atr ), |
| dc.title.none.fl_str_mv | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| dc.type.none.fl_str_mv | Image Figure info:eu-repo/semantics/publishedVersion image |
| description | <p>(A) Representative images acquired by a LSM880 Zeiss confocal microscope from Z stack images of mycATRΔC-/- cells (cl8) untreated (NT) or treated with 5 mM of HU for 5 hours. Previously, cells were incubated with 150 µM of IdU for 16 hours for DNA labelling. Cells were fixed with 3% formaldehyde and stained with α-BrdU (ssDNA, green); genomic DNA was stained using DAPI (gray). Dashed squared delimitated a cell zoomed out. (n) nucleus (k) kinetoplast are indicated. Bar scale = 8 um. (B) Quantification of the ssDNA signal in the nucleus of mycATR and mycATRΔC-/- cells treated or not with 5 mM of HU for 5 hours; Cas9T7 cells without α-BrdU were use as negative control. An area was drawn around 150 nuclei based on DAPI staining and ssDNA signal was measured using ImageJ software (Error bars ± SD; n = 2 experiments, p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001. Unpaired t-test). (C) mycATR and mycATRΔC-/- cells were left untreated (NT) or treated with 0.5 mM of Hydroxyurea (HU) for 20 hours (Chronic) or treated with 5 mM of Hydroxyurea (HU) for 5 hours (Acute), washed and resuspended in fresh media, and samples collected at 0, 2, 5 hours after chronic treatment and 0, 2, 4 hours after acute treatment. Cells were fixed with methanol and DNA stained with propidium iodate, followed by flow cytometer analysis; the cell cycle profile was created using FlowJo software. (D) Representative images of mycATRΔC-/- (cl1) cells pulsed with 150 µM of EdU for 30 minutes in untreated condition or after 5 hours culturing in fresh media post release from chronic HU treatment. Cells were fixed with 3% formaldehyde and a Click-IT reaction was used to stain cells that had uptaken EdU during the 30 min pulse (red); genomic DNA was stained with DAPI (cyan) and α-tubulin was used to stain the cytoskeleton (gray). Dashed squared delimitated a cell zoomed out. (n) nucleus (k) kinetoplast are indicated. Bar = 6 um. (E) Percentage of EdU positive mycATR or mycATRΔC-/- cells in each condition (untreated, after release from chronic (post 5 hours), or acute (post 4 hours) HU treatment) was calculated by the number of positive EdU stained cells divided by the total number of cells present in each sample (100 – 300 cells; error bars ± SD; n = 2 experiments, * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001. Unpaired t-test).</p> |
| eu_rights_str_mv | openAccess |
| id | Manara_a736a7c556eca71d418b189fd950c0e0 |
| identifier_str_mv | 10.1371/journal.pgen.1011899.g003 |
| network_acronym_str | Manara |
| network_name_str | ManaraRepo |
| oai_identifier_str | oai:figshare.com:article/30697536 |
| publishDate | 2025 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells.Gabriel L. A. da Silva (22676649)Jeziel D. Damasceno (9073663)Jennifer A. Black (14411790)Craig Lapsley (6082646)Richard McCulloch (12918)Luiz R. O. Tosi (9073666)BiophysicsBiochemistryMicrobiologyCell BiologyGeneticsMolecular BiologyDevelopmental BiologyScience PolicyInfectious DiseasesBiological Sciences not elsewhere classifiedPhysical Sciences not elsewhere classifiedmajor &# 8217div >< pdata reveals losscells possess mechanismsremarkably plastic genomecas9 genome editinglike family actsprotein kinase ataxiaatr depends upondelayed dna replicationstranded dna accumulationleishmania major </dna damage kinasedna damagegenome stabilityleishmania </dna metabolismdna injuriesatr actsreplication stressreplication impedimentswork showswhose integrityterminal knockoutsmaller chromosomesprotozoan parasitephosphatidylinositol 3nuclear localisationmycatrδc -/-).mycatr ),master regulatorlarger chromosomeskey roleincreased levelsfunctional relevanceeukaryotic responsedeletion resultsconstantly challengedatr playsatr homologatr ),<p>(A) Representative images acquired by a LSM880 Zeiss confocal microscope from Z stack images of mycATRΔC-/- cells (cl8) untreated (NT) or treated with 5 mM of HU for 5 hours. Previously, cells were incubated with 150 µM of IdU for 16 hours for DNA labelling. Cells were fixed with 3% formaldehyde and stained with α-BrdU (ssDNA, green); genomic DNA was stained using DAPI (gray). Dashed squared delimitated a cell zoomed out. (n) nucleus (k) kinetoplast are indicated. Bar scale = 8 um. (B) Quantification of the ssDNA signal in the nucleus of mycATR and mycATRΔC-/- cells treated or not with 5 mM of HU for 5 hours; Cas9T7 cells without α-BrdU were use as negative control. An area was drawn around 150 nuclei based on DAPI staining and ssDNA signal was measured using ImageJ software (Error bars ± SD; n = 2 experiments, p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001. Unpaired t-test). (C) mycATR and mycATRΔC-/- cells were left untreated (NT) or treated with 0.5 mM of Hydroxyurea (HU) for 20 hours (Chronic) or treated with 5 mM of Hydroxyurea (HU) for 5 hours (Acute), washed and resuspended in fresh media, and samples collected at 0, 2, 5 hours after chronic treatment and 0, 2, 4 hours after acute treatment. Cells were fixed with methanol and DNA stained with propidium iodate, followed by flow cytometer analysis; the cell cycle profile was created using FlowJo software. (D) Representative images of mycATRΔC-/- (cl1) cells pulsed with 150 µM of EdU for 30 minutes in untreated condition or after 5 hours culturing in fresh media post release from chronic HU treatment. Cells were fixed with 3% formaldehyde and a Click-IT reaction was used to stain cells that had uptaken EdU during the 30 min pulse (red); genomic DNA was stained with DAPI (cyan) and α-tubulin was used to stain the cytoskeleton (gray). Dashed squared delimitated a cell zoomed out. (n) nucleus (k) kinetoplast are indicated. Bar = 6 um. (E) Percentage of EdU positive mycATR or mycATRΔC-/- cells in each condition (untreated, after release from chronic (post 5 hours), or acute (post 4 hours) HU treatment) was calculated by the number of positive EdU stained cells divided by the total number of cells present in each sample (100 – 300 cells; error bars ± SD; n = 2 experiments, * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001. Unpaired t-test).</p>2025-11-24T18:33:13ZImageFigureinfo:eu-repo/semantics/publishedVersionimage10.1371/journal.pgen.1011899.g003https://figshare.com/articles/figure/Replication_stress_leads_to_ssDNA_accumulation_which_affects_the_mechanisms_influencing_cell_cycle_progression_in_mycATR_C-_-_cells_/30697536CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/306975362025-11-24T18:33:13Z |
| spellingShingle | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. Gabriel L. A. da Silva (22676649) Biophysics Biochemistry Microbiology Cell Biology Genetics Molecular Biology Developmental Biology Science Policy Infectious Diseases Biological Sciences not elsewhere classified Physical Sciences not elsewhere classified major &# 8217 div >< p data reveals loss cells possess mechanisms remarkably plastic genome cas9 genome editing like family acts protein kinase ataxia atr depends upon delayed dna replication stranded dna accumulation leishmania major </ dna damage kinase dna damage genome stability leishmania </ dna metabolism dna injuries atr acts replication stress replication impediments work shows whose integrity terminal knockout smaller chromosomes protozoan parasite phosphatidylinositol 3 nuclear localisation mycatrδc -/-). mycatr ), master regulator larger chromosomes key role increased levels functional relevance eukaryotic response deletion results constantly challenged atr plays atr homolog atr ), |
| status_str | publishedVersion |
| title | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| title_full | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| title_fullStr | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| title_full_unstemmed | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| title_short | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| title_sort | Replication stress leads to ssDNA accumulation, which affects the mechanisms influencing cell cycle progression in mycATRΔC-/- cells. |
| topic | Biophysics Biochemistry Microbiology Cell Biology Genetics Molecular Biology Developmental Biology Science Policy Infectious Diseases Biological Sciences not elsewhere classified Physical Sciences not elsewhere classified major &# 8217 div >< p data reveals loss cells possess mechanisms remarkably plastic genome cas9 genome editing like family acts protein kinase ataxia atr depends upon delayed dna replication stranded dna accumulation leishmania major </ dna damage kinase dna damage genome stability leishmania </ dna metabolism dna injuries atr acts replication stress replication impediments work shows whose integrity terminal knockout smaller chromosomes protozoan parasite phosphatidylinositol 3 nuclear localisation mycatrδc -/-). mycatr ), master regulator larger chromosomes key role increased levels functional relevance eukaryotic response deletion results constantly challenged atr plays atr homolog atr ), |