Journal: bioRxiv

Article Title: Deep learning enables fast and dense single-molecule localization with high accuracy

doi: 10.1101/2020.10.26.355164

Figure Lengend Snippet: a) DECODE can reduce acquisition times by one order of magnitude. The same sample of microtubules, labeled with anti- α tubulin primary and AF647 secondary antibodies, imaged with different UV activation intensities to result in different emitter densities between 0.08 and 0.86 emitters per frame per μm 2 and acquisition times between 93 and 1120 s, while keeping the total number of localizations the same. For high-density activation, we show a comparison with CSpline. b) Fourier Ring Correlation curves for DECODE and CSpline for different emitter densities. c) Resolution estimates obtained using the Fourier Ring Correlation and 0.143 criterion across densities for both methods. d) Fast live-cell SMLM on the nuclear pore complex protein Nup96-mMaple acquired in 3 seconds. e) DECODE enables ultra-high labeling densities. Microtubules labeled with a high concentration of anti- α and anti- β tubulin primary and AF647 secondary antibodies. e1, e2) Magnified regions as indicated in a. Data acquired with high-density labeling shows continuous structures. As a comparison, the same sample was acquired after pre-bleaching of the fluorophores to reach the single-molecule blinking regime. Here, single labels are resolved in the superresolution reconstruction and lead to a sparse decoration of the microtubules. e3, e4) Side view reconstructions of regions as indicated in e1, e2 resolving the hollow, cylinder-like structure of immunolabeled microtubules. f) Representative raw camera frames for the high-density and single-emitter acquisitions, respectively. Scale bars: 10 μm (d inset, f), 1 μm (a, d, e, e1, e2), 100 nm (e3,e4).

Article Snippet: For imaging of live cells, coverslips containing Nup96-mMaple cells (catalog no. 300461, CLS Cell Line Service, Eppelheim, Germany) were rinsed twice with warm PBS before they were mounted onto a custom manufactured sample holder in 1 mL growth medium containing 20 mM HEPES buffer and imaged directly.

Techniques: Labeling, Activation Assay, Concentration Assay, Immunolabeling

Journal: Clinical Cancer Research

Article Title: Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

doi: 10.1158/1078-0432.CCR-22-2977

Figure Lengend Snippet: The Polθ inhibitor ART558 radiosensitizes tumor cells. A and B, Clonogenic survival of HCT116, H460, and T24 cells treated with ART558 and/or IR. A, Plating efficiency for unirradiated cells. B, Surviving fractions as a function of the irradiation dose. Representative wells for 0 Gy and 6 Gy ± 1 μmol/L ART558 are shown for each cell line. C, Clonogenic survival of U2OS WT and Polθ KO cells treated with 3 μmol/L ART558 and 6 Gy IR. Bar graphs show the surviving fraction at 6 Gy. The Western blot insets confirm the lack of Polθ expression in the U2OS Polθ KO cells. D, Clonogenic survival of HCT116 and H460 cells transfected with either a control, nontargeting siRNA (siNT) or an siRNA targeted against POLQ (si POLQ ) and treated with 3 μmol/L ART558 and 6 Gy IR. Bar graphs show the surviving fraction at 6 Gy. Western blots show Polθ expression at the time of irradiation. E, Clonogenic survival of H460 and HCT116 treated with 3 μmol/L ART558 and 5×2 Gy (2 Gy once per day for 1 to 5 days). F and G, Clonogenic survival following irradiation and treatment with 3 μmol/L ART558 of synchronized HeLa cells. F, Representative histograms showing the cell-cycle distribution at the time of IR (synchronized in G 1 by DT block or after 6 hours release from DT block, compared with asynchronous cultures). G, Degree of radiosensitization estimated by the ratio between the surviving fraction of DMSO- and ART558-treated cells after IR (SF DMSO / SF ART558). Data correspond to average ± SD from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: U2OS Polθ KO cells were generated CRISPR/Cas9 technology by Synthego for Artios, which also provided the U2OS WT cells (RRID:CVCL_0042).

Techniques: Irradiation, Western Blot, Expressing, Transfection, Control, Blocking Assay

Journal: Clinical Cancer Research

Article Title: Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

doi: 10.1158/1078-0432.CCR-22-2977

Figure Lengend Snippet: Characterization of ART899 as a specific and potent Polθ inhibitor with improved stability. A, Chemical structures of the Polθ inhibitors ART558 and ART899. The table shows the in vitro intrinsic clearance values of ART558 and ART899 after exposure to rat and mouse liver microsomes. B, Nano-luciferase MMEJ assay showing ART899-mediated inhibition of MMEJ activity in HEK-293 cells. The nano-luciferase readings were normalized to control luciferase (firefly) readings, and these were then normalized to DMSO. Data points show the mean ± SEM of four technical replicates; representative of two independent experiments. C, Confirmation of MMEJ assay specificity. Same experiment described in B but showing both the nanoluc and firefly readings normalized to their own DMSO reading, confirming negligible inhibition by ART899 of the control firefly luciferase signal. D, Clonogenic survival of HCT116 and H460 cells treated with ART899. Graphs show the surviving fraction after 5 × 2 Gy IR. E, Confirmation of ART899 specificity in U2OS WT and Polθ KO cells. Cells were treated as described in D . F, Effect of ART899 in noncancerous cells. MRC-5 and AG01552 fibroblasts were treated as described in D . The effect of ART899 in unirradiated cells from D to F is shown in Supplementary Fig. S5A. Graphs shown in D to F correspond to average ± SD from triplicate wells (representative from three separate experiments; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). G, Viability of HIEC-6 cells treated with ART899 and irradiated with 5 × 2 Gy, as determined by the alamar blue assay 8 days after the first IR fraction. Graph shows the viability normalized to unirradiated controls (Supplementary Fig. S5C); representative from three independent experiments.

Article Snippet: U2OS Polθ KO cells were generated CRISPR/Cas9 technology by Synthego for Artios, which also provided the U2OS WT cells (RRID:CVCL_0042).

Techniques: In Vitro, Luciferase, Inhibition, Activity Assay, Control, Irradiation, Alamar Blue Assay

Journal: Clinical Cancer Research

Article Title: Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models

doi: 10.1158/1078-0432.CCR-22-2977

Figure Lengend Snippet: Polθ inhibitor ART899 combined with radiation causes significant tumor growth delay in vivo and is well tolerated. A, ART899 plasma concentration following oral dosage of ART899 at 50 or 150 mg/kg. Mouse plasma samples ( n = 3 per treatment group) were collected at 30 minutes, 1, 2, 4, 8, and 12 hours after last dose. B–E, HCT116 tumor-bearing mice treated with 150 mg/kg Polθ inhibitor ART899 twice daily for 12 days and/or 10 × 2 Gy (days 1–5 and 8–12). Vehicle ( n = 9); ART899 ( n = 10); 10 × 2 Gy + vehicle ( n = 10); 10 × 2 Gy + ART899 ( n = 10). B, Mean ± SEM relative tumor size. P value from mixed effect model and Dunnett post-test. Comparison of tumor size at the latest common timepoint for 10 × 2 Gy versus 10 × 2 Gy + ART899 are shown in Supplementary Fig. S6A. C, Individual mouse graphs. D, Kaplan–Meier plot for a tumor size threshold of 1,000 mm 3 . HR: Hazard ratio (hazard rate of IR arm / hazard rate of IR + ART899 arm); P value from the log-rank (Mantel-Cox) test comparing IR alone and IR + ART558. The median time to a tumor size of 1,000 mm 3 for the IR + ART899 arm versus the IR arm and the corresponding ratio are shown in Supplementary Fig. S6B. E, Average mouse weight ± SD from all treatment groups over time. Individual mouse weights are shown in Supplementary Fig. S6C.

Article Snippet: U2OS Polθ KO cells were generated CRISPR/Cas9 technology by Synthego for Artios, which also provided the U2OS WT cells (RRID:CVCL_0042).

Techniques: In Vivo, Clinical Proteomics, Concentration Assay, Comparison

Journal: iScience

Article Title: YAP condensates are highly organized hubs

doi: 10.1016/j.isci.2024.109927

Figure Lengend Snippet:

Article Snippet: The YAP–HaloTag CRISPR knock-in U-2 OS cells were plated into eight-well LabTek chambered coverglass dishes (life technologies, 155409PK) for drug treatment and imaging the following day.

Techniques: Virus, Recombinant, Modification, Protease Inhibitor, Transfection, Reverse Transcription, SYBR Green Assay, Hybridization, Single Particle, CRISPR, Knock-In, Negative Control, Plasmid Preparation, Software