Journal: Cell death & disease

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns.

doi: 10.1038/cddis.2014.450

Figure Lengend Snippet: Figure 7 Calpain activity is a prerequisite for the release of nucleosomes and DAMPs in dying cells. HeLa cells were incubated in amino acid-depleted medium (HBSS) or staurosporine-containing medium (Stau) (1 μg/ml) for 24 or 8 h, respectively, in the presence of solvent control, BAPTA-AM (50 μM) or A23187 (1 μg/ml). Released DNA was quantified with PicoGreen (a). Amount of intracellular free Ca2+ was measured in cells treated with solvent control or staurosporine at the indicated time periods by eFluor 514 (b). Cells were treated with staurosporine for 8 h in the presence of control solvent, ALLN (20 μM), or PD150606 (20 μM), and released DNA or protein was detected by PicoGreen staining (c) or western blot for histones and DAMPs (d), respectively. Genomic DNA was separated in agarose gel electrophoresis from staurosporine-treated cells with or without PD150606 (e). The cells treated with staurosporine and PD150606 for 8 h, were stained for lamin A/C and histone H1, and examined by confocal microscopy; the arrows indicate nuclear fragmentations (f). Cells treated with control solvent, zVAD-fmk (20 μM), or PD150606 with or without staurosporine for 8 h were measured for viabilities with Calcein assay (g). Cells treated with staurosporine were measured for calpain activity, caspases 3 and 7 activity, or released DNAs at the indicated time periods (h). HeLa cells transfected with non- targeting shRNA (NT), calpastatin shRNA, calpain S1 shRNA, or overexpressing calpain 1, calpain 2, or calpastatin were incubated with staurosporine for 8 h and the released DNAs were measured by PicoGreen staining (i). Cells expressing calpain 1 or calpain 2 tagged with Flag at the N-terminus and HA at the C-terminus were treated with staurosporine and western-blotted with anti-Flag antibody or anti-HA antibody (j). HeLa cells expressing halide-sensitive YFP were treated with staurosporine and/or PD150606 for 4 h, and their chloride ion currents were measured by fluorometry by detecting quenching of YFP fluorescence (k). Data performed in triplicate are presented as mean± S.D. (a–c and g–i)

Article Snippet: Cl-amidine was purchased from Cayman Chemical (Ann Arbor, MI, USA). zVAD-fmk, zDEVD-fmk, Necrostatin-1, BAPTA-AM, diphenyliodonium, PD150606, ALLN and A23187 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Incubation, Solvent, Control, Staining, Western Blot, Agarose Gel Electrophoresis, Confocal Microscopy, Transfection, shRNA, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Capturing Translation in Action with Protein Synthesis Profiling

doi: 10.1101/2025.11.17.688896

Figure Lengend Snippet: a, Structural model of APEX2-eEF2 bound to the ribosome. The large ribosomal subunit is shown in cyan, the small subunit in green, APEX2-eEF2 in magenta, and mRNA as a black line with the red circles indicating the alkyne modification. The blue sphere marks the ∼25 nm labeling radius of APEX2. b, Expression constructs for APEX2 and APEX2-eEF2 under the TDH3 promoter. c, Western blot detection of APEX2 and APEX2-eEF2 expression. Lanes: control (empty vector), APEX2 (27 kDa), and APEX2-eEF2 (118 kDa). Tubulin (Tub) was probed as a loading control. d, Schematic of the RNA tagging workflow. Yeast cells were incubated with alkyne-phenol (30 min), followed by H₂O₂ (5 min). After quenching, total RNA was extracted, conjugated to biotin-azide via click chemistry, and enriched with streptavidin beads. Both total and enriched RNA were used for preparing Illumina sequencing libraries. e, Agarose gel analysis of total RNA from control, APEX2, and APEX2-eEF2 cells. The presence of intact 25S and 18S rRNA bands indicates high RNA quality. M, molecular weight ladder. f, Detection of alkyne-labeled RNAs by conjugation with fluorescein-azide. Total RNA from control, APEX2, and APEX2-eEF2 cells was subjected to click chemistry and analyzed by agarose gel electrophoresis. Fluorescence was detected using a Typhoon imager. g, The same gel as in F, stained with SafeStain to verify equal RNA loading. h, Quantification of fluorescein-labeled RNA signal. The bar graph shows fluorescence intensity normalized to total RNA, averaged across two independent experiments. i, Gel-shift assay of biotin-labeled RNAs incubated with anti-biotin-AF488 antibody. RNAs from control, APEX2, and APEX2-eEF2 cells were conjugated with biotin-azide, bound by antibody, and resolved on an agarose gel. Antibody-RNA complexes are indicated by the black bar. Lanes: M, molecular weight ladder; control, RNA from control cells; APEX2, RNA from APEX2-expressing cells; APEX2-eEF2, RNA from APEX2-eEF2-expressing cells; Ab, antibody only.

Article Snippet: After incubation, RNA was purified using the Monarch RNA Cleanup Kit (New England BioLabs, Cat# T2040) according to the manufacturer’s instructions.

Techniques: Modification, Labeling, Expressing, Construct, Western Blot, Control, Plasmid Preparation, Incubation, Illumina Sequencing, Agarose Gel Electrophoresis, Molecular Weight, Conjugation Assay, Fluorescence, Staining, Gel Shift