wild type u2os cells  (ATCC)

Journal: Environmental and Molecular Mutagenesis

Article Title: Oxidative DNA Damage Exacerbates the Mutagenic Potential of Alternative DNA Structures via Altered DNA Repair Processing

doi: 10.1002/em.70059

Figure Lengend Snippet: Mutation frequencies of B‐DNA and H‐DNA sequences with or without oxidative damage and XPA, a key NER protein. B‐DNA‐ or H‐DNA‐forming reporter sequences were exposed to different levels of oxidative stress (−, +, or ++) and then transfected into human U2OS cell lines with either (A and B) wild type or (C and D) XPA knockout phenotypes to allow for DNA repair processing. Mutation frequencies were quantified (A and C) using a blue‐white screening assay. Each condition represents the average of at least three replicates (+SEM). Statistical comparisons between conditions were performed using a two‐way ANOVA with significance indicated as p < 0.05 (*), or < 0.01 (**). Mutation spectra were then determined (B and D) using Sanger sequencing, with at least 15 mutants analyzed per condition and classified as point mutations (1 bp), small deletions (< 15 bp), or large deletions (> 15 bp). Mutation spectra ratios are shown per condition as a ratio of mutation frequency.

Article Snippet: Wild‐type U2OS cells (ATCC, #HTB‐96) were used unless otherwise specified, including the CRISPR‐Cas9 knockout U2OS cell lines for either OGG1, APE1, or XPA (provided by Dr. Robert Sobol, Brown University) and previously characterized (Bordelon ).

Techniques: Mutagenesis, Transfection, Knock-Out, Screening Assay, Sequencing

Journal: Environmental and Molecular Mutagenesis

Article Title: Oxidative DNA Damage Exacerbates the Mutagenic Potential of Alternative DNA Structures via Altered DNA Repair Processing

doi: 10.1002/em.70059

Figure Lengend Snippet: Mutation frequencies of B‐DNA and H‐DNA sequences with or without oxidative damage and key BER proteins. B‐DNA‐ or H‐DNA‐forming reporter sequences were exposed to increasing levels of oxidative stress (−, +, or ++) and then transfected into human U2OS cell lines with either (A and B) OGG1 knockout or (C and D) APE1 knockout phenotypes to allow for DNA repair processing. Mutation frequencies were then quantified (A and C) using a blue‐white screening assay. Each condition represents the average of at least three replicates (+SEM). Statistical comparisons between conditions were performed using a two‐way ANOVA with significance indicated as p < 0.05 (*), < 0.01 (**), or < 0.001 (***). Mutation spectra were then determined (B and D) using Sanger sequencing, with at least 15 mutants analyzed per condition and classified as point mutations (1 bp), small deletions (< 15 bp), or large deletions (> 15 bp). Mutation spectra ratios are shown per condition as a ratio of mutation frequency.

Article Snippet: Wild‐type U2OS cells (ATCC, #HTB‐96) were used unless otherwise specified, including the CRISPR‐Cas9 knockout U2OS cell lines for either OGG1, APE1, or XPA (provided by Dr. Robert Sobol, Brown University) and previously characterized (Bordelon ).

Techniques: Mutagenesis, Transfection, Knock-Out, Screening Assay, Sequencing

Journal: Environmental and Molecular Mutagenesis

Article Title: Oxidative DNA Damage Exacerbates the Mutagenic Potential of Alternative DNA Structures via Altered DNA Repair Processing

doi: 10.1002/em.70059

Figure Lengend Snippet: Association of XPA and APE1 proteins with B‐DNA or H‐DNA sequences with or without oxidative damage and key BER or NER proteins. B‐DNA‐ or H‐DNA‐forming reporter sequences were first exposed to increasing levels of oxidative stress (−, +, or ++). Reporter sequences were then transfected into human U2OS cell lines with (A) wild‐type (previously published matched control (Zewail‐Foote et al. )), (B) XPA knockout, or (C) APE1 knockout phenotypes for DNA repair processing. Chromatin immunoprecipitation (ChIP) was then used to measure the association of XPA or APE1 proteins with B‐DNA or H‐DNA sequences. Protein association is shown as a percentage of total input DNA (% input). Each condition shows the average % input of three replicates (+SEM). Condition comparisons use a Wilcoxon rank sums approach demonstrating p < 0.05 (*), or < 0.01 (**).

Article Snippet: Wild‐type U2OS cells (ATCC, #HTB‐96) were used unless otherwise specified, including the CRISPR‐Cas9 knockout U2OS cell lines for either OGG1, APE1, or XPA (provided by Dr. Robert Sobol, Brown University) and previously characterized (Bordelon ).

Techniques: Transfection, Control, Knock-Out, Chromatin Immunoprecipitation

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: A subset of microtubules has an expanded lattice in cells. (A) Tomogram slice (thickness: 10 nm) showing two selected microtubule (MT) backbones in an untreated U2OS cell (red and pink). (B and C) Power spectra of the masked and transformed MT segments from the red MT (B) and pink MT (C) shown in A. (D) Overlay of the layer line plots of the power spectra of the MT segments from the compacted (red) and expanded (pink) MTs in A. Arrows indicate the location of the layer line peaks and their related lattice spacing. (E) Violin plot showing the distribution of lattice spacings in untreated U2OS cells ( N = 31, 12 tomograms, 7 cells), from microtubules assembled in vitro from GTP-bound soluble tubulin yielding dynamic microtubules ( N = 40, 6 tomograms), in the presence of Taxol ( N = 32, 3 tomograms), or from GMPCPP-bound soluble tubulin ( N = 33, 14 tomograms). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. (F) Simplified cartoon showing the long-range effect of a compacted or an expanded MT lattice. Scale bar: 100 nm (A).

Article Snippet: U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zurich, Zürich, Switzerland).

Techniques: Transformation Assay, In Vitro

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Taxol treatment induces a hyperexpanded lattice within cells. (A) Tomogram slice showing a representative image of Taxol-treated microtubules in WT U2OS cells. Scale bar: 100 nm. (B) Violin plot showing the lattice spacing distribution in Taxol treated cells ( N = 30, 6 tomograms, 5 cells) and in untreated cells ( N = 31, 12 tomograms, 7 cells, same data as , included for comparison). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. Taxol distribution is significantly different from the untreated distribution (****P value <0.0001, unpaired t test based permutation test). (C) Microtubule average shows that Taxol-treated microtubules consist of 13 PFs. Central volume slices (28 nm thick) from top (left) and side (right) views. Scale bar: 5 nm.

Article Snippet: U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zurich, Zürich, Switzerland).

Techniques: Comparison

Journal: The Journal of Cell Biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Correlation of FM to SEM data using an integrated cryo-FM. (A) Cartoon describing the FM-SEM correlation with FM data obtained after milling. Correlation is confirmed using extracellular beads. (B) FIB image of an intact U2OS cell (9° tilted side view). (C) Untilted SEM image of the same grid square as shown in B. The beads used to confirm FM-SEM correlation are indicated with white and black arrows in C and D, respectively. (D) Untilted SEM image of the polished lamella of the cell shown in C. (E) Scatterplot of correlation errors from leave-one-out calculations; each dataset has a unique color, grey circles mark the 1xSD and 2xSD boundaries (10 datasets, 51 beads), dx = difference in x, dy = difference in y. (F) Boxplot showing the distribution of scaling factors (mean = 0.584, standard deviation = 0.005, N = 10). (G) Scaled FM image of the extracellular beads used to guide FM-SEM overlay, beads used to confirm FM-SEM correlation are indicated with black arrows, similar to C and D. (H) Scaled FM image of fBSA-Au 5 beads used for subsequent FM-TEM correlation (see ). (I) Scaled FM image of the StableMARK signal. Scalebars: 10 µm (B–D and G–I).

Article Snippet: U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Zurich, Zürich, Switzerland).

Techniques: Standard Deviation

Journal: The Journal of cell biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices.

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Figure 1. A subset of microtubules has an expanded lattice in cells. (A) Tomogram slice (thickness: 10 nm) showing two selected microtubule (MT) backbones in an untreated U2OS cell (red and pink). (B and C) Power spectra of the masked and transformed MT segments from the red MT (B) and pink MT (C) shown in A. (D) Overlay of the layer line plots of the power spectra of the MT segments from the compacted (red) and expanded (pink) MTs in A. Arrows indicate the location of the layer line peaks and their related lattice spacing. (E) Violin plot showing the distribution of lattice spacings in untreated U2OS cells (N = 31, 12 tomograms, 7 cells), from microtubules assembled in vitro from GTP-bound soluble tubulin yielding dynamic microtubules (N = 40, 6 tomograms), in the presence of Taxol (N = 32, 3 tomograms), or from GMPCPP-bound soluble tubulin (N = 33, 14 tomograms). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. (F) Simplified cartoon showing the long-range effect of a compacted or an expanded MT lattice. Scale bar: 100 nm (A).

Article Snippet: Cell lines and cell culture U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Figure 4.

Techniques: Transformation Assay, In Vitro

Journal: The Journal of cell biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices.

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Figure 2. Taxol treatment induces a hyperexpanded lattice within cells. (A) Tomogram slice showing a representative image of Taxol-treated micro- tubules in WT U2OS cells. Scale bar: 100 nm. (B) Violin plot showing the lattice spacing distribution in Taxol treated cells (N = 30, 6 tomograms, 5 cells) and in untreated cells (N = 31, 12 tomograms, 7 cells, same data as Fig. 1 E, included for comparison). Horizontal lines correspond to the discrete spatial frequency values in reciprocal space. Taxol distribution is significantly different from the untreated distribution (****P value <0.0001, unpaired t test based permutation test). (C) Microtubule average shows that Taxol-treated microtubules consist of 13 PFs. Central volume slices (28 nm thick) from top (left) and side (right) views. Scale bar: 5 nm.

Article Snippet: Cell lines and cell culture U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Figure 4.

Techniques: Comparison

Journal: The Journal of cell biology

Article Title: StableMARK-decorated microtubules in cells have expanded lattices.

doi: 10.1083/jcb.202206143

Figure Lengend Snippet: Figure 3. Correlation of FM to SEM data using an integrated cryo-FM. (A) Cartoon describing the FM-SEM correlation with FM data obtained after milling. Correlation is confirmed using extracellular beads. (B) FIB image of an intact U2OS cell (9° tilted side view). (C) Untilted SEM image of the same grid square as shown in B. The beads used to confirm FM-SEM correlation are indicated with white and black arrows in C and D, respectively. (D) Untilted SEM image of the polished lamella of the cell shown in C. (E) Scatterplot of correlation errors from leave-one-out calculations; each dataset has a unique color, grey circles mark the 1xSD and 2xSD boundaries (10 datasets, 51 beads), dx = difference in x, dy = difference in y. (F) Boxplot showing the distribution of scaling factors (mean = 0.584, standard deviation = 0.005, N = 10). (G) Scaled FM image of the extracellular beads used to guide FM-SEM overlay, beads used to confirm FM-SEM correlation are indicated with black arrows, similar to C and D. (H) Scaled FM image of fBSA-Au5 beads used for subsequent FM-TEM correlation (see Fig. 4). (I) Scaled FM image of the StableMARK signal. Scalebars: 10 µm (B–D and G–I).

Article Snippet: Cell lines and cell culture U2OS wild-type (WT) cells were purchased from ATCC and U2OS Flp-In T-Rex cells were a kind gift from Prof. Alessandro Sartori (Institute of Molecular Cancer Research, University of Figure 4.

Techniques: Standard Deviation

Journal: Open biology

Article Title: A novel FAM83G variant from palmoplantar keratoderma patient disrupts WNT signalling via loss of FAM83G-CK1α interaction.

doi: 10.1098/rsob.240075

Figure Lengend Snippet: Figure 5. Reduced WNT signalling activity in skin fibroblasts derived from the PPK patient. (a) WT U2OS, control fibroblasts (FIB03) and PPK patient skin fibroblasts (FIB04) were incubated with L-CM or Wnt3A-CM for 6 h before RNA extraction, cDNA synthesis and analysis of Axin2 and GAPDH transcripts by qRT-PCR. Axin2 mRNA expression was normalized to GAPDH and the mean of three biological replicates was plotted as a bar graph ± s.d. (b) WT U2OS, control fibroblasts (FIB15, FIB03) and patient skin fibroblasts (FIB04) were incubated with L-CM and Wnt3A-CM for 6 h before lysis. Extracts (20 µg protein) were resolved by SDS-PAGE and transferred to PVDF membranes, which were analysed by immunoblotting using the indicated antibodies. (c) As in (a), except the cells were pre-treated with DMSO or the GSK3 inhibitor CHIR99021 (GSK3i) at 5 µM for 6 h before RNA extraction, cDNA synthesis and qRT-PCR. (d) WT U2OS, control fibroblasts (FIB03), patient skin fibroblasts (FIB04) and two PPK patient cell lines retrovirally transduced to overexpress either WT FAM83G or CK1-binding deficient mutant FAM83GF296A were incubated with L-CM or Wnt3A-CM for 6 h and processed as in (a) for qRT-PCR to measure normalized Axin2 mRNA expression. Statistical analysis was performed using two-way ANOVA with multiple comparisons, **p < 0.01, ns = no statistical significance (n = 3, error bars represent ± s.d.).

Article Snippet: Cell culture Wild-type (WT) U2OS (HTB−96; ATCC), WT DLD1 (CCL−221; ATCC), FAM83G−/− DLD1 [19] and HEK293FT cells (Invitrogen, R70007) were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) FBS (Thermo Fisher 2 royalsocietypublishing.org/journal/rsob Open Biol.

Techniques: Activity Assay, Derivative Assay, Control, Incubation, RNA Extraction, cDNA Synthesis, Quantitative RT-PCR, Expressing, Lysis, SDS Page, Western Blot, Binding Assay, Mutagenesis