Journal: bioRxiv

Article Title: Unscheduled DNA replication in G1 causes genome instability through head-to-tail replication fork collisions

doi: 10.1101/2021.09.06.459115

Figure Lengend Snippet: (A) Summary of engineered genetic changes that allow to bypass cell cycle control of DNA replication and induce unscheduled replication in G1. Indicated proteins and variants are expressed from pGAL1-10 promoter inducible by galactose. Experimental setup for G1 replication involves G1 cell cycle arrest using 10 µg/ml α -factor in 2% raffinose medium, followed by induction of G1 replication by 2% galactose. (B) Bypassing CDK control (Dpb11, Sld2-T84D) and CDK/DDK control (Dbf4, Dpb11, Sld2-T84D) generates different levels of unscheduled replication in G1. Cy5-labeled EdU incorporated after induction of replication in G1-arrested cells and SYTOX green-stained total DNA content were measured by flow cytometry at indicated timepoints. (C) Unscheduled replication in G1 after bypass of CDK or CDK/DDK control occurs genome-wide. EdU-labeled DNA as a proxy for DNA synthesis was isolated after 3 h of G1 replication and mapped to all sixteen S. cerevisiae chromosomes. Data from n=2 replicates. (D) and (E) Unscheduled G1 replication initiates at canonical replication origins. (D) Input-normalized coverage of 60 kb windows around early-replicating replication origins (autonomous replicating sequences (ARS)) with EdU-labeled DNA after 3 h replication in G1 in the presence of 60 mM hydroxyurea (HU) separated into two clusters based on mean signal intensity. (top) Profile plots of mean coverage (dark) ± SE (light). (bottom) Heatmaps with 2.5 Kb bin size. Data from n=2 replicates. (E) Representative EdU-IP coverage traces from the same experiment spanning the entire chromosome 4. Dotted lines indicate early-replicating ARSs. See also .

Article Snippet: One half was subjected to a click chemistry reaction with disulfo-Cy5-picolyl-azide (Jena Bioscience CLK-1177) for one hour, whereas the other half was kept as a control.

Techniques: Labeling, Staining, Flow Cytometry, Genome Wide, DNA Synthesis, Isolation

Journal: bioRxiv

Article Title: Unscheduled DNA replication in G1 causes genome instability through head-to-tail replication fork collisions

doi: 10.1101/2021.09.06.459115

Figure Lengend Snippet: (A) DNA content increases linearly during unscheduled replication in G1. Related to . Mean fluorescence intensity of total DNA (SYTOX green, blue) was measured by flow cytometry after induction of G1 replication for the indicated amount of time. Data were corrected for background mitochondrial DNA synthesis in a control strain, normalized to a DNA content of 1 C and fitted with a linear regression model (see equation). (B) DNA is quantitatively labeled with EdU during G1 replication. Related to . As in (A), additionally measuring mean fluorescence intensity of newly synthesized DNA in G1 (EdU-Cy5, turquoise). Data were corrected for background mitochondrial DNA synthesis in a control strain and normalized to the respective maximum value. (C) Early-replicating origins are activated during G1 replication. Related to . Input-normalized EdU-sequencing data were analyzed at early or late replication origins (ARS) ± 30 Kb. (top) Profile plots of mean coverage (dark) ± SE (light). (bottom) Heatmaps with 1 Kb bin size. Data from n=2 replicates.

Article Snippet: One half was subjected to a click chemistry reaction with disulfo-Cy5-picolyl-azide (Jena Bioscience CLK-1177) for one hour, whereas the other half was kept as a control.

Techniques: Fluorescence, Flow Cytometry, DNA Synthesis, Labeling, Synthesized, Sequencing

Journal: bioRxiv

Article Title: Unscheduled DNA replication in G1 causes genome instability through head-to-tail replication fork collisions

doi: 10.1101/2021.09.06.459115

Figure Lengend Snippet: (A) Split-Venus tagged Dpb11- and Sld2-constructs are expressed to similar levels. Expression levels of Sld2 and Dpb11 carrying split-Venus tags as well as phosphorylated Rad53 detected by western blots from log-phase samples. Note the faint signal for phosphorylated Rad53 with constructs that stabilize the physical interaction between Dpb11 and Sld2. (B) Split-Venus tags (VN/VC) stabilize the physical interaction between Dpb11 and Sld2. Dpb11-VC and Sld2 tagged at either N- or C-terminus with VN fragment of the fluorescent protein Venus. Data represents the resulting mean (light green) and 97 th percentile (dark green) of split-Venus fluorescence intensity measured by flow cytometry in log-phase cells from n=6 replicates. (C) Venus-stabilized interaction of Dpb11 and Sld2 induces DNA replication in G1. Cells of the indicated genotypes were pre-arrested in G1 for 1 h and then kept arrested in G1 in the presence of EdU. Incorporated EdU was labeled with Cy5 and measured by flow cytometry. Note the logarithmic scaling of the x-axis to resolve small amounts of G1 replication. (D) Venus-stabilized interaction of Dpb11 and Sld2 results in cell cycle arrest. Additional samples to experiment shown in . SYTOX green-stained total DNA from samples at the indicated timepoints after release from G1 arrest to the next G1-phase as measured by flow cytometry. (E) Sporadic G1 replication affects long chromosomes more strongly. RPA asymmetry scores for each chromosome were calculated by normalizing the log2-ratios of RPA-ChIP-seq reads mapping to forward and reverse strand in 50 bp bins for chromosome length. Data from n=2 replicates. (F) Long chromosomes harbor more early-firing origins. Length of chromosomes was plotted against the total number of ARS sequences. Color intensity indicates the number of early-firing origins. (G) High levels of genome instability are caused by Venus-stabilized interaction of Dpb11 and Sld2. GCR rates for the assay shown in were calculated from n=8 cultures by fluctuation analysis. Error bars indicate a 95% confidence interval for the determined GCR rate. Note the logarithmic scaling of the y-axis.

Article Snippet: One half was subjected to a click chemistry reaction with disulfo-Cy5-picolyl-azide (Jena Bioscience CLK-1177) for one hour, whereas the other half was kept as a control.

Techniques: Construct, Expressing, Western Blot, Fluorescence, Flow Cytometry, Labeling, Staining, ChIP-sequencing

Journal: Theranostics

Article Title: In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates

doi: 10.7150/thno.16003

Figure Lengend Snippet: (a) Schematic illustration of Ac 4 ManDBCO-mediated metabolic labeling of cancer cells and subsequent detection of cell-surface DBCO groups using Cy5-azide via copper-free Click chemistry. (b) CLSM images of LS174T cells after treatment with Ac 4 ManDBCO (20 μM) or PBS for three days and further incubation with Cy5-azide (20 μM) for 30 min. Cell nuclei were stained with DAPI (blue). Scale bar represents 10 μm. (c) Flow cytometry analyses of LS174T cells following the same treatment in (b). (d) Mean Cy5 fluorescence intensity of LS174T cells extracted from (b). Data were presented as mean ± SEM (n=3) and analyzed by Student's t-test (two-tailed) (0.01 < *P ≤ 0.05, and 0.001 < **P ≤ 0.01).

Article Snippet: Cy5-azide was purchased from Kerafast (Boston, MA, USA).

Techniques: Labeling, Incubation, Staining, Flow Cytometry, Fluorescence, Two Tailed Test

Journal: Theranostics

Article Title: In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates

doi: 10.7150/thno.16003

Figure Lengend Snippet: (a) Schematic illustration of the preparation of azido-/Cy5-NCs and 64 Cu-/azido-/Cy5-NCs. The chemical structures of TEOS, DOTA-silane, Cy5-silane, and azido-PEG 3.4k -silane were shown. Cy5-NCs and 64 Cu-/Cy5-NCs were synthesized similarly except that azido-PEG 3.4k -silane was replaced with mPEG 3.4k -silane. (b) SEM image of azido-/Cy5-NCs showed a size of 48.8 ± 4.9 nm. Scale bar represents 200 nm. (c) Fluorescence spectra of azido-/Cy5-NCs and Cy5-NCs. The excitation wavelength was set at 650 nm. (d) FTIR spectra of silica core, azido-/Cy5-NCs and Cy5-NCs. Inset: enlarged view of transmittance profiles of azido-/Cy5-NCs and Cy5-NCs within the wavenumber range of 2095-2114 cm -1 . (e) Loss of 64 Cu over time from 64 Cu-/azido-/Cy5-NCs or 64 Cu-/Cy5-NCs following incubation at 37 o C in 10% FBS (n=3).

Article Snippet: Cy5-azide was purchased from Kerafast (Boston, MA, USA).

Techniques: Synthesized, Fluorescence, Incubation

Journal: Theranostics

Article Title: In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates

doi: 10.7150/thno.16003

Figure Lengend Snippet: (a) Schematic illustration of the targeted and non-targeted internalization of azido-/Cy5-NCs by cancer cells. (b) Flow cytometry profiles of LS174T cells following treatment with either Ac 4 ManDBCO (blue curve) or PBS (red curve) for 72 h and further incubation with azido-/Cy5-NCs for 30 min. Cells without azido-/Cy5-NCs treatment served as negative controls (black curve). (c) Mean fluorescence intensity of LS174T cells after incubation with azido-/Cy5-NCs or Cy5-NCs for 0.5, 1, and 2 h, respectively following pre-treatment with Ac 4 ManDBCO for 72 h. Cells treated with azido-/Cy5-NCs following pretreatment with PBS served as controls (n=6). Statistically significantly lower Cy5 fluorescence intensity than Ac 4 ManDBCO+azido-/Cy5-NCs group was observed in both PBS+azido-/Cy5-NCs and Ac 4 ManDBCO+Cy5-NCs groups (0.01 < * P ≤ 0.05, and 0.001 < ** P ≤ 0.01).

Article Snippet: Cy5-azide was purchased from Kerafast (Boston, MA, USA).

Techniques: Flow Cytometry, Incubation, Fluorescence

Journal: Theranostics

Article Title: In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates

doi: 10.7150/thno.16003

Figure Lengend Snippet: (a) Time frame of in vivo imaging study. LS174T tumors were implanted in both flanks of athymic nude mice. When the tumors reached ~50 mm 3 , Ac 4 ManDBCO (5 mg/kg) was intratumorally injected into the left tumors once daily for three days (Day 1-3), while the right tumors were injected with PBS as controls. On Day 4, 64 Cu-/azido-/Cy5-NCs or 64 Cu-/Cy5-NCs (~100 μCi) were i.v. injected and their biodistribution were monitored via PET/CT imaging. (b) PET/CT imaging of athymic nude mouse at 24 h post injection of 64 Cu-/azido-/Cy5-NCs. The tumor area was zoomed in to clearly show the retention of 64 Cu-/azido-/Cy5-NCs in the left and right tumors. Tumors were shown by the arrows. Inset: calculated radioactivity of the left and right tumors using the imaging software. (c) Biodistribution of 64 Cu-/azido-/Cy5-NCs and 64 Cu-/Cy5-NCs at 24 h post injection, as determined by ex vivo radioactivity measurement on a λ-counter. Data were presented as mean ± SEM (n=3) and analyzed by Student's t-test (two-tailed) (0.001 < **P ≤ 0.01).

Article Snippet: Cy5-azide was purchased from Kerafast (Boston, MA, USA).

Techniques: In Vivo Imaging, Injection, Positron Emission Tomography-Computed Tomography, Imaging, Radioactivity, Software, Ex Vivo, Two Tailed Test

Journal: Theranostics

Article Title: In Vivo Targeting of Metabolically Labeled Cancers with Ultra-Small Silica Nanoconjugates

doi: 10.7150/thno.16003

Figure Lengend Snippet: (a) Whole-body fluorescence imaging of athymic nude mice bearing LS174T tumors at 1, 6, 24, 48, and 72 h post injection of azido-/Cy5-NCs. Tumors were shown by the arrows. The left tumors were injected with Ac 4 ManDBCO (5 mg/kg) once daily for three days (Day 1-3), while the right tumors were injected with PBS as controls. Azido-/Cy5-NCs or Cy5-NCs were i.v. injected on Day 4. (b) Ex vivo imaging of tissues harvested at 72 h post injection of azido-/Cy5-NCs. (c) Mean Cy5 fluorescence intensity of tissues extracted from ex vivo images (n=3). (d) Representative CLSM images of tissue sections of the left and right tumors from mice treated with azido-/Cy5-NCs. Cell nuclei were stained with DAPI (blue).

Article Snippet: Cy5-azide was purchased from Kerafast (Boston, MA, USA).

Techniques: Fluorescence, Imaging, Injection, Ex Vivo, Staining