research cell line source s human osteosarcoma u2os cells Search Results


cell lines cell line source s u2os  (ATCC)
98
ATCC cell lines cell line source s u2os
Cell Lines Cell Line Source S U2os, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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reference identifiers additional information cell line human u2os atcc htb 96 cell line  (ATCC)
99
ATCC reference identifiers additional information cell line human u2os atcc htb 96 cell line
Figure 1. Protein aggregation is increased following a functional loss of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and upon topoisomerase poisoning. See also Figure 1—figure supplement 1. (A) In-gel Coomassie staining of indicated fractions of cell extracts of WT and ATM KO <t>U2OS</t> cells. The relative amounts of each fraction loaded are indicated. (B) Quantification of (A). Circles depict individual experiments; gray dotted lines depict matched pairs. Wilcoxon matched-pairs signed-rank test. (C) Aggregated (silver stain) and whole-cell lysate (WCL; Coomassie) fractions of HEK293T cells treated transiently with chemical agents targeting the indicating proteins (see Table 1 for drugs and doses used; for etoposide [Etop]: 3 μM; for camptothecin [CPT]: 100 nM). See also Figure 1—figure supplement 1D. (D) Quantification of (C). Circles depict individual experiments. Two-tailed Student’s t-test with Bonferroni correction. (E) Protein fractions of HEK293T cells treated transiently with increasing amounts of CPT (20–100 nM) or Etop (0.6–3 μM). (F) Quantification of (E). Two-tailed Student’s t-test with Bonferroni correction. (G) Protein fractions of HEK293T cells treated transiently with CPT (40 nM) or Etop (1.5 μM), targeting TOP1 or TOP2, respectively, 24 hr or 72 hr after treatment. (H) Quantification of (G). Two-tailed Student’s t-test with Bonferroni correction. In (B), (D), (F), and (H), the red line indicates the mean.
Reference Identifiers Additional Information Cell Line Human U2os Atcc Htb 96 Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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u2os human bone sarcoma cell line  (ATCC)
99
ATCC u2os human bone sarcoma cell line
Figure 1. Protein aggregation is increased following a functional loss of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and upon topoisomerase poisoning. See also Figure 1—figure supplement 1. (A) In-gel Coomassie staining of indicated fractions of cell extracts of WT and ATM KO <t>U2OS</t> cells. The relative amounts of each fraction loaded are indicated. (B) Quantification of (A). Circles depict individual experiments; gray dotted lines depict matched pairs. Wilcoxon matched-pairs signed-rank test. (C) Aggregated (silver stain) and whole-cell lysate (WCL; Coomassie) fractions of HEK293T cells treated transiently with chemical agents targeting the indicating proteins (see Table 1 for drugs and doses used; for etoposide [Etop]: 3 μM; for camptothecin [CPT]: 100 nM). See also Figure 1—figure supplement 1D. (D) Quantification of (C). Circles depict individual experiments. Two-tailed Student’s t-test with Bonferroni correction. (E) Protein fractions of HEK293T cells treated transiently with increasing amounts of CPT (20–100 nM) or Etop (0.6–3 μM). (F) Quantification of (E). Two-tailed Student’s t-test with Bonferroni correction. (G) Protein fractions of HEK293T cells treated transiently with CPT (40 nM) or Etop (1.5 μM), targeting TOP1 or TOP2, respectively, 24 hr or 72 hr after treatment. (H) Quantification of (G). Two-tailed Student’s t-test with Bonferroni correction. In (B), (D), (F), and (H), the red line indicates the mean.
U2os Human Bone Sarcoma Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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u2os cells  (Human Protein Atlas)
86
Human Protein Atlas u2os cells
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
U2os Cells, supplied by Human Protein Atlas, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cell lines source identifier 293t atcc hela atcc u2os atcc hct116 fbxw7  (ATCC)
99
ATCC cell lines source identifier 293t atcc hela atcc u2os atcc hct116 fbxw7
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Cell Lines Source Identifier 293t Atcc Hela Atcc U2os Atcc Hct116 Fbxw7, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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be-c  (ATCC)
96
ATCC be-c
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Be C, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sk-n-as  (ATCC)
96
ATCC sk-n-as
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Sk N As, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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sk-n-sh  (ATCC)
96
ATCC sk-n-sh
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Sk N Sh, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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u-2 os cells  (AstraZeneca ltd)
90
AstraZeneca ltd u-2 os cells
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
U 2 Os Cells, supplied by AstraZeneca ltd, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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vero c1008  (ATCC)
99
ATCC vero c1008
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Vero C1008, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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datasheet  (Cell Signaling Technology Inc)
97
Cell Signaling Technology Inc datasheet
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Datasheet, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human osteosarcoma saos-2 cell line  (Merck KGaA)
90
Merck KGaA human osteosarcoma saos-2 cell line
a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of <t>U2OS</t> cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).
Human Osteosarcoma Saos 2 Cell Line, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Figure 1. Protein aggregation is increased following a functional loss of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and upon topoisomerase poisoning. See also Figure 1—figure supplement 1. (A) In-gel Coomassie staining of indicated fractions of cell extracts of WT and ATM KO U2OS cells. The relative amounts of each fraction loaded are indicated. (B) Quantification of (A). Circles depict individual experiments; gray dotted lines depict matched pairs. Wilcoxon matched-pairs signed-rank test. (C) Aggregated (silver stain) and whole-cell lysate (WCL; Coomassie) fractions of HEK293T cells treated transiently with chemical agents targeting the indicating proteins (see Table 1 for drugs and doses used; for etoposide [Etop]: 3 μM; for camptothecin [CPT]: 100 nM). See also Figure 1—figure supplement 1D. (D) Quantification of (C). Circles depict individual experiments. Two-tailed Student’s t-test with Bonferroni correction. (E) Protein fractions of HEK293T cells treated transiently with increasing amounts of CPT (20–100 nM) or Etop (0.6–3 μM). (F) Quantification of (E). Two-tailed Student’s t-test with Bonferroni correction. (G) Protein fractions of HEK293T cells treated transiently with CPT (40 nM) or Etop (1.5 μM), targeting TOP1 or TOP2, respectively, 24 hr or 72 hr after treatment. (H) Quantification of (G). Two-tailed Student’s t-test with Bonferroni correction. In (B), (D), (F), and (H), the red line indicates the mean.

Journal: eLife

Article Title: Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome

doi: 10.7554/elife.70726

Figure Lengend Snippet: Figure 1. Protein aggregation is increased following a functional loss of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and upon topoisomerase poisoning. See also Figure 1—figure supplement 1. (A) In-gel Coomassie staining of indicated fractions of cell extracts of WT and ATM KO U2OS cells. The relative amounts of each fraction loaded are indicated. (B) Quantification of (A). Circles depict individual experiments; gray dotted lines depict matched pairs. Wilcoxon matched-pairs signed-rank test. (C) Aggregated (silver stain) and whole-cell lysate (WCL; Coomassie) fractions of HEK293T cells treated transiently with chemical agents targeting the indicating proteins (see Table 1 for drugs and doses used; for etoposide [Etop]: 3 μM; for camptothecin [CPT]: 100 nM). See also Figure 1—figure supplement 1D. (D) Quantification of (C). Circles depict individual experiments. Two-tailed Student’s t-test with Bonferroni correction. (E) Protein fractions of HEK293T cells treated transiently with increasing amounts of CPT (20–100 nM) or Etop (0.6–3 μM). (F) Quantification of (E). Two-tailed Student’s t-test with Bonferroni correction. (G) Protein fractions of HEK293T cells treated transiently with CPT (40 nM) or Etop (1.5 μM), targeting TOP1 or TOP2, respectively, 24 hr or 72 hr after treatment. (H) Quantification of (G). Two-tailed Student’s t-test with Bonferroni correction. In (B), (D), (F), and (H), the red line indicates the mean.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70726 18 of 29 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) U2OS ATCC HTB- 96 Cell line (human) U2OS ATM KO This study See ‘Mammalian cell culture’ Cell line (human) U2OS + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) U2OS ATM KO + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) Phoenix- Ampho ATCC RRID:CVCL_H716 Retrovirus packaging cell line Antibody GFP (mouse, monoclonal) Takara Bio Clontech 632380 WB (1:5000) Antibody ATM (mouse, monoclonal) Santa Cruz Sc- 23921 WB (1:200) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 159 WB (1:2000) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 165 IF (1:200) Antibody GAPDH (mouse, monoclonal) Fitzgerald 10R- G109a WB (1:10,000) Antibody TUB (mouse, monoclonal) Sigma- Aldrich T5138 WB (1:4000) Antibody HDAC1 (mouse, monoclonal) DSHB PCPR- HDAC1- 2E12 WB (0.5 μg/ml) Antibody MCM7 (Mmouse, monoclonal) Santa Cruz 47DC141 WB (1:100) Antibody TUBA1A (mouse, monoclonal) Sigma- Aldrich T5168 WB (1:2000) Antibody FUS (mouse, monoclonal) Santa Cruz Sc- 47711 IF (1:200) Antibody 53BP1 (rabbit, monoclonal) Santa Cruz Sc- 22760 IF (1:150) Antibody 53BP1 (rabbit, monoclonal) Bethyl A300- 272A IF (1:500) Antibody HSP70 (mouse, monoclonal) StressMarq SMC- 104A IF (1:100) Antibody HSP90 (mouse, monoclonal) StressMarq SMC- 149 IF (1:100) Recombinant DNA reagent pQCXIN–HSPB5 (plasmid) PMID:20843828 Recombinant DNA reagent ATM CRISPR/Cas9 KO (plasmid) Santa Cruz sc- 400192 Recombinant DNA reagent ATM HDR (plasmid) Santa Cruz sc- 400192- HDR Sequence- based reagent HEK293Q71F (forward primer) This study PCR primer GAGTCC CTCAAG TCCTTCC Sequence- based reagent HEK293Q71R (reverse primer) This study PCR primer AAACGG GCCCTC TAGACTC Commercial assay or kit Silver stain kit Pierce (Thermo Scientific) 24612 Commercial assay or kit Allprep DNA/RNA isolation mini kit QIAGEN 80004 Commercial assay or kit S- trap micro Protifi K02- micro- 10 Commercial assay or kit Masterpure Complete DNA and RNA purificiation kit Epicentre (supplied through Lucigen) MC85200 Commercial assay or kit QuantSeq 3’ mRNA- Seq library prep kit (FWD) Lexogen 015.96 Chemical compound, drug Camptothecin Selleckchem S1288 See Table 1 Chemical compound, drug Etoposide Sigma- Aldrich E1383 See Table 1 Chemical compound, drug TDP1 inhibitor Merck 532177 See Table 1 Continued Continued on next page Huiting et al. eLife 2022;11:e70726.

Techniques: Functional Assay, Staining, Silver Staining, Two Tailed Test

Figure 2. Camptothecin (CPT) and ataxia telangiectasia mutated (ATM) loss drives aggregation in a cell-type-dependent manner. See also Figure 2— figure supplement 1. (A) Volcano plot of label-free quantification (LFQ) MS/MS analysis of the aggregated fractions of DMSO and CPT-treated HEK293T cells. n = 4. Only proteins identified in >1 repeats of either case or control are shown. (B) Volcano plot of LFQ MS/MS analysis of the aggregated fractions of DMSO and ATM inhibitor-treated HEK293T cells. n = 4. Only proteins identified in >1 repeats of either case or control are shown. (C) Venn diagram showing overlap between U2OS and HEK293T increased aggregation, after the indicated treatments. (D) Western blot using the indicated antibodies on the aggregated and whole-cell lysate (WCL) fractions of drug-treated and ATM KO HEK293 cells, and wild-type U2OS cells. n = 2. (E) GO term analysis (Function) of the increased aggregation in CPT- or ATM-inhibitor-treated HEK293T cells. (F) Venn diagram showing overlap between increased aggregation after the indicated treatments in HEK293T cells and baseline aggregation in U2OS cells. (G) Aggregated (silver stain) and WCL (Coomassie) fractions of untreated HEK293T and U2OS cells. n = 2.

Journal: eLife

Article Title: Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome

doi: 10.7554/elife.70726

Figure Lengend Snippet: Figure 2. Camptothecin (CPT) and ataxia telangiectasia mutated (ATM) loss drives aggregation in a cell-type-dependent manner. See also Figure 2— figure supplement 1. (A) Volcano plot of label-free quantification (LFQ) MS/MS analysis of the aggregated fractions of DMSO and CPT-treated HEK293T cells. n = 4. Only proteins identified in >1 repeats of either case or control are shown. (B) Volcano plot of LFQ MS/MS analysis of the aggregated fractions of DMSO and ATM inhibitor-treated HEK293T cells. n = 4. Only proteins identified in >1 repeats of either case or control are shown. (C) Venn diagram showing overlap between U2OS and HEK293T increased aggregation, after the indicated treatments. (D) Western blot using the indicated antibodies on the aggregated and whole-cell lysate (WCL) fractions of drug-treated and ATM KO HEK293 cells, and wild-type U2OS cells. n = 2. (E) GO term analysis (Function) of the increased aggregation in CPT- or ATM-inhibitor-treated HEK293T cells. (F) Venn diagram showing overlap between increased aggregation after the indicated treatments in HEK293T cells and baseline aggregation in U2OS cells. (G) Aggregated (silver stain) and WCL (Coomassie) fractions of untreated HEK293T and U2OS cells. n = 2.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70726 18 of 29 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) U2OS ATCC HTB- 96 Cell line (human) U2OS ATM KO This study See ‘Mammalian cell culture’ Cell line (human) U2OS + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) U2OS ATM KO + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) Phoenix- Ampho ATCC RRID:CVCL_H716 Retrovirus packaging cell line Antibody GFP (mouse, monoclonal) Takara Bio Clontech 632380 WB (1:5000) Antibody ATM (mouse, monoclonal) Santa Cruz Sc- 23921 WB (1:200) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 159 WB (1:2000) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 165 IF (1:200) Antibody GAPDH (mouse, monoclonal) Fitzgerald 10R- G109a WB (1:10,000) Antibody TUB (mouse, monoclonal) Sigma- Aldrich T5138 WB (1:4000) Antibody HDAC1 (mouse, monoclonal) DSHB PCPR- HDAC1- 2E12 WB (0.5 μg/ml) Antibody MCM7 (Mmouse, monoclonal) Santa Cruz 47DC141 WB (1:100) Antibody TUBA1A (mouse, monoclonal) Sigma- Aldrich T5168 WB (1:2000) Antibody FUS (mouse, monoclonal) Santa Cruz Sc- 47711 IF (1:200) Antibody 53BP1 (rabbit, monoclonal) Santa Cruz Sc- 22760 IF (1:150) Antibody 53BP1 (rabbit, monoclonal) Bethyl A300- 272A IF (1:500) Antibody HSP70 (mouse, monoclonal) StressMarq SMC- 104A IF (1:100) Antibody HSP90 (mouse, monoclonal) StressMarq SMC- 149 IF (1:100) Recombinant DNA reagent pQCXIN–HSPB5 (plasmid) PMID:20843828 Recombinant DNA reagent ATM CRISPR/Cas9 KO (plasmid) Santa Cruz sc- 400192 Recombinant DNA reagent ATM HDR (plasmid) Santa Cruz sc- 400192- HDR Sequence- based reagent HEK293Q71F (forward primer) This study PCR primer GAGTCC CTCAAG TCCTTCC Sequence- based reagent HEK293Q71R (reverse primer) This study PCR primer AAACGG GCCCTC TAGACTC Commercial assay or kit Silver stain kit Pierce (Thermo Scientific) 24612 Commercial assay or kit Allprep DNA/RNA isolation mini kit QIAGEN 80004 Commercial assay or kit S- trap micro Protifi K02- micro- 10 Commercial assay or kit Masterpure Complete DNA and RNA purificiation kit Epicentre (supplied through Lucigen) MC85200 Commercial assay or kit QuantSeq 3’ mRNA- Seq library prep kit (FWD) Lexogen 015.96 Chemical compound, drug Camptothecin Selleckchem S1288 See Table 1 Chemical compound, drug Etoposide Sigma- Aldrich E1383 See Table 1 Chemical compound, drug TDP1 inhibitor Merck 532177 See Table 1 Continued Continued on next page Huiting et al. eLife 2022;11:e70726.

Techniques: Quantitative Proteomics, Tandem Mass Spectroscopy, Control, Western Blot, Silver Staining

Figure 5. The lowered aggregation threshold caused by topoisomerase poisoning or a loss of ataxia telangiectasia mutated (ATM) is accompanied by a rewiring and aggregation of known interacting (co)chaperones. See also Figure 5—figure supplements 1 and 2. (A) Presence of (co)chaperones in the aggregated protein fractions in HEK293T cells. Left panel: Venn diagram showing the overlap in aggregating chaperones. Right panel: graph depicting the overlap in aggregating chaperones in detail. (B) Pie charts showing the presence of HSPA1A and HSPA8 clients in aggregated protein fractions compared to clients present in both NIA fractions. See text for reference; only clients identified in at least two out of three repeats in Lee et al. were taken into account here. (C) Table showing the number of (co)chaperones logged in BioGRID as interacting with NIA and aggregating protein fractions. (D) See also (A): only (co)chaperones aggregating in both HEK293T experimental set-ups or significantly more in one of them are shown here. BioGRID (co)chaperone interactions with the aggregated proteins identified in this study, per (co)chaperone. Darker colors represent a higher percentage of proteins with a reported binding to that (co)chaperone. See Figure 5—figure supplement 2 for a complete overview. (E) Volcano plot showing protein levels of (co)chaperones in camptothecin (CPT)-treated HEK293T cells compared to DMSO-treated cells. (F) Differentially expressed (co)chaperones in CPT-treated HEK293T cells compared to DMSO-treated cells based on RNAseq data. (G) Western blot analysis using the indicated antibodies on the whole-cell lysate (WCL) fractions of HEK293T cells after the indicated time and treatment. HS, heat shock (2 hr at 43°C incubator). n = 2. (H) Graph showing (co)chaperones whose genes show a differential expression in both CPT-treated HEK293T cells compared to DMSO-treated HEK293T cells and in untreated U2OS compared to untreated HEK293T cells.

Journal: eLife

Article Title: Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome

doi: 10.7554/elife.70726

Figure Lengend Snippet: Figure 5. The lowered aggregation threshold caused by topoisomerase poisoning or a loss of ataxia telangiectasia mutated (ATM) is accompanied by a rewiring and aggregation of known interacting (co)chaperones. See also Figure 5—figure supplements 1 and 2. (A) Presence of (co)chaperones in the aggregated protein fractions in HEK293T cells. Left panel: Venn diagram showing the overlap in aggregating chaperones. Right panel: graph depicting the overlap in aggregating chaperones in detail. (B) Pie charts showing the presence of HSPA1A and HSPA8 clients in aggregated protein fractions compared to clients present in both NIA fractions. See text for reference; only clients identified in at least two out of three repeats in Lee et al. were taken into account here. (C) Table showing the number of (co)chaperones logged in BioGRID as interacting with NIA and aggregating protein fractions. (D) See also (A): only (co)chaperones aggregating in both HEK293T experimental set-ups or significantly more in one of them are shown here. BioGRID (co)chaperone interactions with the aggregated proteins identified in this study, per (co)chaperone. Darker colors represent a higher percentage of proteins with a reported binding to that (co)chaperone. See Figure 5—figure supplement 2 for a complete overview. (E) Volcano plot showing protein levels of (co)chaperones in camptothecin (CPT)-treated HEK293T cells compared to DMSO-treated cells. (F) Differentially expressed (co)chaperones in CPT-treated HEK293T cells compared to DMSO-treated cells based on RNAseq data. (G) Western blot analysis using the indicated antibodies on the whole-cell lysate (WCL) fractions of HEK293T cells after the indicated time and treatment. HS, heat shock (2 hr at 43°C incubator). n = 2. (H) Graph showing (co)chaperones whose genes show a differential expression in both CPT-treated HEK293T cells compared to DMSO-treated HEK293T cells and in untreated U2OS compared to untreated HEK293T cells.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70726 18 of 29 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) U2OS ATCC HTB- 96 Cell line (human) U2OS ATM KO This study See ‘Mammalian cell culture’ Cell line (human) U2OS + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) U2OS ATM KO + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) Phoenix- Ampho ATCC RRID:CVCL_H716 Retrovirus packaging cell line Antibody GFP (mouse, monoclonal) Takara Bio Clontech 632380 WB (1:5000) Antibody ATM (mouse, monoclonal) Santa Cruz Sc- 23921 WB (1:200) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 159 WB (1:2000) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 165 IF (1:200) Antibody GAPDH (mouse, monoclonal) Fitzgerald 10R- G109a WB (1:10,000) Antibody TUB (mouse, monoclonal) Sigma- Aldrich T5138 WB (1:4000) Antibody HDAC1 (mouse, monoclonal) DSHB PCPR- HDAC1- 2E12 WB (0.5 μg/ml) Antibody MCM7 (Mmouse, monoclonal) Santa Cruz 47DC141 WB (1:100) Antibody TUBA1A (mouse, monoclonal) Sigma- Aldrich T5168 WB (1:2000) Antibody FUS (mouse, monoclonal) Santa Cruz Sc- 47711 IF (1:200) Antibody 53BP1 (rabbit, monoclonal) Santa Cruz Sc- 22760 IF (1:150) Antibody 53BP1 (rabbit, monoclonal) Bethyl A300- 272A IF (1:500) Antibody HSP70 (mouse, monoclonal) StressMarq SMC- 104A IF (1:100) Antibody HSP90 (mouse, monoclonal) StressMarq SMC- 149 IF (1:100) Recombinant DNA reagent pQCXIN–HSPB5 (plasmid) PMID:20843828 Recombinant DNA reagent ATM CRISPR/Cas9 KO (plasmid) Santa Cruz sc- 400192 Recombinant DNA reagent ATM HDR (plasmid) Santa Cruz sc- 400192- HDR Sequence- based reagent HEK293Q71F (forward primer) This study PCR primer GAGTCC CTCAAG TCCTTCC Sequence- based reagent HEK293Q71R (reverse primer) This study PCR primer AAACGG GCCCTC TAGACTC Commercial assay or kit Silver stain kit Pierce (Thermo Scientific) 24612 Commercial assay or kit Allprep DNA/RNA isolation mini kit QIAGEN 80004 Commercial assay or kit S- trap micro Protifi K02- micro- 10 Commercial assay or kit Masterpure Complete DNA and RNA purificiation kit Epicentre (supplied through Lucigen) MC85200 Commercial assay or kit QuantSeq 3’ mRNA- Seq library prep kit (FWD) Lexogen 015.96 Chemical compound, drug Camptothecin Selleckchem S1288 See Table 1 Chemical compound, drug Etoposide Sigma- Aldrich E1383 See Table 1 Chemical compound, drug TDP1 inhibitor Merck 532177 See Table 1 Continued Continued on next page Huiting et al. eLife 2022;11:e70726.

Techniques: Binding Assay, Western Blot, Quantitative Proteomics

Figure 6. Protein aggregation triggered by genotoxic stress is amenable to modulation by chaperones. See also Figure 6—figure supplements 1 and 2. (A) Western blot of whole-cell lysate (WCL) and aggregated proteins isolated from HEK293T cells treated transiently with DMSO or camptothecin (CPT), followed by treatment with the VER-155008 HSP70 inhibitor (10 μM), probed with the indicated antibodies. n = 3. (B) Aggregated (silver stain) and WCL (Coomassie) fractions of U2OS wild-type and ATM KO cells, with or without overexpression of HSPB5. (C) Quantification of (B). (D) Representative immunofluorescence pictures of U2OS wild-type and ATM KO cells stably overexpressing HSPB5 or not, stained with ProteoStat (red) and Hoechst (blue). (E) Quantification of aggresome signatures in (D). (F). Representative immunofluorescence pictures of U2OS wild-type and ATM KO cells stably overexpressing HSPB5 or not, stained with anti-FUS (green) and Hoechst (blue). (G) Quantification of extranuclear FUS inclusions in (F). (H) Aggregated (silver stain) and WCL (Coomassie) fractions of HEK293 cells stably overexpressing HSPB5 or not, treated transiently with DMSO or CPT. Three technical repeats are shown here. (I) Quantification of three independent biological repeats of (H). In (C), (E), (G), and (I), squares represent independent biological repeats, bars represent mean ± SEM. p-Values are obtained by two-tailed Student’s t-tests followed by a Bonferroni correction for multiple comparisons.

Journal: eLife

Article Title: Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome

doi: 10.7554/elife.70726

Figure Lengend Snippet: Figure 6. Protein aggregation triggered by genotoxic stress is amenable to modulation by chaperones. See also Figure 6—figure supplements 1 and 2. (A) Western blot of whole-cell lysate (WCL) and aggregated proteins isolated from HEK293T cells treated transiently with DMSO or camptothecin (CPT), followed by treatment with the VER-155008 HSP70 inhibitor (10 μM), probed with the indicated antibodies. n = 3. (B) Aggregated (silver stain) and WCL (Coomassie) fractions of U2OS wild-type and ATM KO cells, with or without overexpression of HSPB5. (C) Quantification of (B). (D) Representative immunofluorescence pictures of U2OS wild-type and ATM KO cells stably overexpressing HSPB5 or not, stained with ProteoStat (red) and Hoechst (blue). (E) Quantification of aggresome signatures in (D). (F). Representative immunofluorescence pictures of U2OS wild-type and ATM KO cells stably overexpressing HSPB5 or not, stained with anti-FUS (green) and Hoechst (blue). (G) Quantification of extranuclear FUS inclusions in (F). (H) Aggregated (silver stain) and WCL (Coomassie) fractions of HEK293 cells stably overexpressing HSPB5 or not, treated transiently with DMSO or CPT. Three technical repeats are shown here. (I) Quantification of three independent biological repeats of (H). In (C), (E), (G), and (I), squares represent independent biological repeats, bars represent mean ± SEM. p-Values are obtained by two-tailed Student’s t-tests followed by a Bonferroni correction for multiple comparisons.

Article Snippet: DOI: https://doi.org/10.7554/eLife.70726 18 of 29 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) U2OS ATCC HTB- 96 Cell line (human) U2OS ATM KO This study See ‘Mammalian cell culture’ Cell line (human) U2OS + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) U2OS ATM KO + HSPB5 This study See ‘Mammalian cell culture’ Cell line (human) Phoenix- Ampho ATCC RRID:CVCL_H716 Retrovirus packaging cell line Antibody GFP (mouse, monoclonal) Takara Bio Clontech 632380 WB (1:5000) Antibody ATM (mouse, monoclonal) Santa Cruz Sc- 23921 WB (1:200) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 159 WB (1:2000) Antibody HSPB5 (mouse, monoclonal) StressMarq SMC- 165 IF (1:200) Antibody GAPDH (mouse, monoclonal) Fitzgerald 10R- G109a WB (1:10,000) Antibody TUB (mouse, monoclonal) Sigma- Aldrich T5138 WB (1:4000) Antibody HDAC1 (mouse, monoclonal) DSHB PCPR- HDAC1- 2E12 WB (0.5 μg/ml) Antibody MCM7 (Mmouse, monoclonal) Santa Cruz 47DC141 WB (1:100) Antibody TUBA1A (mouse, monoclonal) Sigma- Aldrich T5168 WB (1:2000) Antibody FUS (mouse, monoclonal) Santa Cruz Sc- 47711 IF (1:200) Antibody 53BP1 (rabbit, monoclonal) Santa Cruz Sc- 22760 IF (1:150) Antibody 53BP1 (rabbit, monoclonal) Bethyl A300- 272A IF (1:500) Antibody HSP70 (mouse, monoclonal) StressMarq SMC- 104A IF (1:100) Antibody HSP90 (mouse, monoclonal) StressMarq SMC- 149 IF (1:100) Recombinant DNA reagent pQCXIN–HSPB5 (plasmid) PMID:20843828 Recombinant DNA reagent ATM CRISPR/Cas9 KO (plasmid) Santa Cruz sc- 400192 Recombinant DNA reagent ATM HDR (plasmid) Santa Cruz sc- 400192- HDR Sequence- based reagent HEK293Q71F (forward primer) This study PCR primer GAGTCC CTCAAG TCCTTCC Sequence- based reagent HEK293Q71R (reverse primer) This study PCR primer AAACGG GCCCTC TAGACTC Commercial assay or kit Silver stain kit Pierce (Thermo Scientific) 24612 Commercial assay or kit Allprep DNA/RNA isolation mini kit QIAGEN 80004 Commercial assay or kit S- trap micro Protifi K02- micro- 10 Commercial assay or kit Masterpure Complete DNA and RNA purificiation kit Epicentre (supplied through Lucigen) MC85200 Commercial assay or kit QuantSeq 3’ mRNA- Seq library prep kit (FWD) Lexogen 015.96 Chemical compound, drug Camptothecin Selleckchem S1288 See Table 1 Chemical compound, drug Etoposide Sigma- Aldrich E1383 See Table 1 Chemical compound, drug TDP1 inhibitor Merck 532177 See Table 1 Continued Continued on next page Huiting et al. eLife 2022;11:e70726.

Techniques: Western Blot, Isolation, Silver Staining, Over Expression, Immunofluorescence, Stable Transfection, Staining, Two Tailed Test

a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of U2OS cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).

Journal: bioRxiv

Article Title: Robust fluorescent labeling and tracking of endogenous non-repetitive genomic loci

doi: 10.1101/2025.08.22.671818

Figure Lengend Snippet: a Simplified cartoon of dSaCas9-ArrayG/N 8X (not shown to scale). b Kymographs of U2OS cells expressing dSaCas9-ArrayG/N 16X ( top ) without sgRNA (None), ( middle ) with a non-targeting/scrambled sgRNA (Scrambled), and ( bottom ) with Alu-targeting sgRNA (Alu). Intensity normalized independently for each image. Scale bars are 1 µm. c Violin plots of the proportion of tracks per cell greater than 2 seconds for each sgRNA condition ( n = 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). d Survival probability of trajectories for each sgRNA condition ( n = 29,500, 52,614, and 400,063 trajectories in 12, 25, and 87 cells for None, Scrambled, and Alu, respectively). e Representative images (single focal planes) of telomeres labeled with dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP. Intensity normalized independently for each image. Scale bars are 5 µm. f Intensity decay of telomeres labeled by the indicated dCas9 tags and imaged at 20 Hz (data shows mean ± std, n = 228, 78, and 103 trajectories in 10 cells each for dSaCas9-ArrayG/N 16X , dSpCas9-ArrayG/N 16X , and dSpCas9-EGFP, respectively). g Mean squared displacement analysis of telomeres labeled with the indicated dCas9 tags and tracked at 20 Hz, shown as violin plots of the fitted ( top ) effective diffusion coefficients, D*, and ( bottom ) anomalous exponents, α ( n = 1276, 884, 265, 294, 34 trajectories across 78, 57, 40, 40, 30 cells, from left to right).

Article Snippet: Plotting the average diffusion coefficient (D*) of six labeled endogenous loci against their reported expression levels in U2OS cells (sourced from the Human Protein Atlas ) revealed no direct correlation (Supplementary Fig. 9).

Techniques: Expressing, Labeling, Diffusion-based Assay