csk buffer  (Thermo Fisher)

Journal: bioRxiv

Article Title: Targeting Noncanonical TEAD Axis to Overcome DNA Repair-Driven Chemoresistance

doi: 10.64898/2026.04.09.717588

Figure Lengend Snippet: (A) Confocal images of endogenous pan-TEAD (green) and YAP/TAZ (red) in U2OS cells transfected with non-targeting control siRNA (siNC, 20 nM) or combined YAP/TAZ siRNA (siYAP 10 nM + siTAZ 10 nM). “+CSK” indicates samples pre-extracted with CSK buffer prior to fixation to enrich chromatin-associated proteins. (B) Quantification of nuclear pan-TEAD and YAP/TAZ fluorescence intensities from (A). Lines indicate medians (siNC: n = 80; siYAP/TAZ: n = 88; +CSK siNC: n = 100; +CSK siYAP/TAZ: n = 105). (C) TEAD4 ChIP-seq profiles comparing wild-type versus YAP/TAZ-knockout (KO) HEK293A cells. Left: Genomic tracks highlighting regions with stable, lost, or gained TEAD4 binding. Right: Average ChIP-seq signal density at TEAD-LOSS regions. (D) Confocal images of HEK293A YAP/TAZ-knockout cells reconstituted with empty vector, FLAG-YAP-5SA, or FLAG-YAP-5SA/S94A. Cells were stained for pan-TEAD (green) and FLAG (red). (E) Quantification of relative nuclear intensities of pan-TEAD and FLAG in cells from (D), normalized to non-extracted (–CSK) controls. Box-and-whisker plots show minimum–maximum ranges (–CSK control: n = 195; +CSK control: n = 58; YAP-5SA: n = 33; YAP-5SA/S94A: n = 13). (F) TEAD4 ChIP-seq signal at the CTGF promoter in YAP/TAZ-knockout HEK293A cells transfected as in (D). Data from two independent experiments. (G) qPCR analysis of canonical Hippo target genes (ANKRD1, CTGF, CYR61) in YAP/TAZ-knockout HEK293A cells expressing empty vector, YAP-5SA, or YAP-5SA/S94A. Expression values were normalized to control (three independent experiments). (H) Recruitment of pan-TEAD and YAP/TAZ to FokI-induced DNA double-strand breaks (DSBs) in U2OS 2-6-5 cells transfected with non-targeting control siRNA (siNC, 20 nM) or combined siYAP (10 nM) and siTAZ (10 nM). (I) Quantification of pan-TEAD and YAP/TAZ fluorescence intensities at FokI-positive regions, normalized to FokI-negative regions. Lines indicate medians (negative, n = 29; siNC, n = 31; siY/T, n = 28). (J) Immunoblot analysis of Myc–TEAD4 immunoprecipitates from chromatin and whole-cell fractions in HEK293A TEAD1/2/4-knockout cells transfected with Myc–TEAD4 and treated with doxorubicin (5 μM, 2 h) or camptothecin (CPT; 1 μM, 2 h). (K) 8×GTIIC luciferase reporter assay measuring YAP/TAZ–TEAD transcriptional activity in HEK293A wild-type cells treated with doxorubicin or etoposide at 1 μM (+) or 3 μM (++) for 8h. Data represent two biological replicates for etoposide and three for doxorubicin. (L) Immunoblot analysis of Myc–TEAD4 immunoprecipitates from chromatin and whole-cell fractions in HEK293A LATS1/2-knockout cells transfected with Myc–TEAD4 and treated with doxorubicin (5 μM, 2 h).

Article Snippet: Cells were incubated on ice for 5 min in CSK buffer (10 mM HEPES pH 7.4, 100 mM NaCl, 300 mM sucrose, 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, 0.2% Triton X-100) supplemented with HaltTM protease and phosphatase inhibitors (Thermo), followed by PBS wash and fixation as above.

Techniques: Transfection, Control, Fluorescence, ChIP-sequencing, Knock-Out, Binding Assay, Plasmid Preparation, Staining, Whisker Assay, Expressing, Western Blot, Luciferase, Reporter Assay, Activity Assay