live cell imaging chamber  (Tokai Hit Co Ltd)

Journal: Oncogene

Article Title: EZH2 PROTACs outperform catalytic inhibitors in prostate cancer by targeting a methylation-independent function of PRC2

doi: 10.1038/s41388-025-03662-z

Figure Lengend Snippet: A A panel of PCa cell lines (LNCaP, C4-2B, LNCaP95, 22Rv1, and VCaP) and a normal prostate epithelial cell line (RWPE) were treated with PROTAC-6272 and EPZ-6438 with increasing doses from nM to µM for 48 h, and half inhibitory concentrations (IC 50 ) of proliferation rates were assayed by cell Titer Glo ( n = 6). B A panel of PCa cell lines was treated with DMSO, PROTAC-6272 (1 µM), and EPZ-6438 (1 µM) and incubated in an IncuCyte live imager chamber; images were taken every 2 h up to 9 days. Cell proliferation rates were analyzed by IncuCyte live imager software ( n = 6). C 22Rv1 cells were subjected to triplicate RNA-seq analysis upon 6 days of treatment with DMSO, PROTAC-6286, PROTAC-6272, and EPZ-6438 (1 µM). Heatmap shows combined differentially expressed genes identified ( | Log2FC | ≥0.585, adjusted p < 0.05) from treatment of PROTAC-6286, EPZ-6438, or PROTAC-6272 relative to DMSO. D VCaP cells were subjected to triplicate RNA-seq analysis upon 6 days of treatment with DMSO, PROTAC-6286, PROTAC-6272, and EPZ-6438. Heatmap shows combined differentially expressed genes identified ( | Log2FC | ≥0.585, adjusted p < 0.05) from treatment of PROTAC-6286, EPZ-6438, or PROTAC-6272 relative to DMSO.

Article Snippet: For the Incucyte assay, cells were seeded in 96-well plates and allowed to stabilize for 24 h. Cells were then treated with 1 μM of DMSO, PROTAC-6272, and EPZ-6438 and incubated in the Incucyte live imaging chamber (Sartorius).

Techniques: Incubation, Software, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Autophosphorylation of the Tousled-like kinases TLK1 and TLK2 regulates recruitment to damaged chromatin via PCNA interaction

doi: 10.1093/nar/gkae1279

Figure Lengend Snippet: Accumulation of TLK1 and 2 is negatively regulated by their kinase activity and dimerization at damaged chromatin. ( A and C ) TLK1 and TLK2 recruitment to sites of DNA damage is repressed by dimerization and kinase activity. U2OS cells were transfected with indicated GFP-TLK1 or TLK2 wildtype and mutants. Laser-induced micro-irradiation and live-cell imaging experiments were conducted using a confocal microscope, denoted line between the white arrows. Representative images of each experimental group at 10 min after damage. Scale bar represents 10 μm. ( B, D ) Quantification of laser-included micro-irradiation as in (A) and (C), respectively. N ≥ 10 cells. Significance was determined by unpaired, two-tailed Student’s t -test and P -values are reported as * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. ( E ) DNA damage kinetics of LC8, TLK1 and TLK2 catalytic inactive mutants using laser-induced micro-irradiation. Scale bar represents 10 μm. ( F ) Kinase activity negatively regulates TLK chromatin loading. HEK293T cells were transfected with the indicated GFP-TLK1 wildtype and catalytic inactive mutants. Cells were harvested 24 h after transfection and fractionated to obtain soluble and chromatin fractions.

Article Snippet: Laser-induced micro-irradiation was performed using a Nikon Ti2 inverted fluorescent microscope and C2+ confocal system with an Okolab live cell imaging chamber attached to a gas-mixer maintained at a temperature of 37°C and 5% CO 2 .

Techniques: Activity Assay, Transfection, Irradiation, Live Cell Imaging, Microscopy, Two Tailed Test