Journal: Frontiers in Oncology

Article Title: Clickable Cisplatin Derivatives as Versatile Tools to Probe the DNA Damage Response to Chemotherapy

doi: 10.3389/fonc.2022.874201

Figure Lengend Snippet: Cisplatin derivatives are clickable and bind DNA repair proteins. (A) Schematic representation of the copper-catalyzed click reaction (CuAAC) in which an azide (purple) and an alkyne (orange) compound were covalently bound to form a triazole conjugate. (B) Visualization of clickable Pt-alkyne-53 with AF488-picolyl azide and Pt-azide-64 with AF488-alkyne in U2OS cells treated for 3 hours with the indicated compounds at 5 μM or 25 μM, respectively (green). DAPI (blue) was used to counterstain nuclei. Vehicle treated cells (DMF) were used as a negative control. Scale bar represents 20 μm. (C) Dot blot of DNA from U2OS cells, immobilized on a nitrocellulose membrane and stained with streptavidin-HRP to detect DNA-bound biotin. Cells were pre-incubated for 5 hours with 100μM Pt-alkyne-53 or Pt-azide-64, followed by fixation and subjected to a CuAAC click reaction with biotin-azide or biotin-alkyne. Vehicle treated cells (DMF), as well as cells exposed to the CuAAC click reaction without the copper catalyst (-CuSO 4 ), were used as negative controls. Methylene blue staining of the nitrocellulose membrane was used to control for loading. (D) Scheme of the experimental approach to pull-down DNA repair proteins that interact with the cisplatin derivatives. (E) Pull-down of Pt-alkyne-53 from U2OS cells treated with 10μM cisplatin or 100μM Pt-alkyne-53 for 12 hours. The CuAAC click reaction was performed with biotin-azide on chromatin fractions and streptavidin beads were used to pull down the Pt-alkyne-53 along with the proteins bound to it. Proteins enriched in chromatin fractions (input) as well as proteins pulled down with the streptavidin beads (eluate) were probed with the indicated antibodies. The arrow indicates the expected size of the FANCD2 protein.

Article Snippet: The CuAAC click reaction mix composition was: 1 μM AF488-picolyl-azide (from kit C10641, Invitrogen) or 5 μM AF488-alkyne (CLK-1277-1, Jena Bioscience), pre-mixed CuSO 4 :THPTA (2 mM CuSO 4 (Jena Bioscience), 4 mM THPTA (762342, Sigma-Aldrich), final concentrations), 10 mM sodium ascorbate (PHR1279, Sigma-Aldrich) diluted in PBS.

Techniques: Negative Control, Dot Blot, Membrane, Staining, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Neodymium-Facilitated Visualization of Extreme Phosphate Accumulation in Fibroblast Filopodia: Implications for Intercellular and Cell–Matrix Interactions

doi: 10.3390/ijms252011076

Figure Lengend Snippet: Keratocytes treated with NdCl 3 solution on a culture plastic surface measuring 260 × 180 square meters. The images were obtained using a scanning electron microscopy (SEM) equipped with a backscattered electron (BSE) detector. The yellow continuous arrows indicate the connected filopodia of two keratocytes, with a clearly visible extreme contrast zone. Yellow dotted arrows indicate single filopodia with a clear zone of extreme contrast perpendicular to them. The question mark is used in cases where it is not possible to speak with certainty about intercellular communication through contact with filopodia: ( a ) Control cell culture: typical, uniformly bright, elongated keratocytes with normally shaped, round nuclei are observed. ( b ) Cell culture with lipopolysaccharide (LPS)-induced apoptosis: the keratocyte membrane exhibits characteristic apoptotic vesicles (some of which are indicated by the red arrows). The red dotted line limits the group of cells that have the most significant apoptotic changes. Keratocytes in this group show a relatively low level of brightness on the SEM-BSE image with neodymium staining. They have become rounded and their nuclei have lost their shape. The number of filopodia with extreme contrast zones, which are associated with an asymmetric increase in brightness, is higher in this group compared to the control group ( a ), particularly at the boundary of the zone with pronounced apoptosis.

Article Snippet: Detection of apoptosis was performed on a flow cytometer BeamCyte 1026M (VDO Biotech, Suzhou, China) after staining for annexin V and propidium iodide (21172 Apoptosis Detection Kit, Lumiprobe RUS Ltd., Moscow, Russia).

Techniques: Electron Microscopy, Control, Cell Culture, Membrane, Staining

Journal: International Journal of Molecular Sciences

Article Title: Neodymium-Facilitated Visualization of Extreme Phosphate Accumulation in Fibroblast Filopodia: Implications for Intercellular and Cell–Matrix Interactions

doi: 10.3390/ijms252011076

Figure Lengend Snippet: Phosphate “cannibalism” in close-up view. The keratocyte on the left is seen pulling Pi through filopodia from a keratocyte on the right that has previously undergone apoptosis. The scanning electron microscopy (SEM) image (in backscattered electron (BSE) detection mode) of keratocytes treated with NdCl 3 solution on culture plastic.

Article Snippet: Detection of apoptosis was performed on a flow cytometer BeamCyte 1026M (VDO Biotech, Suzhou, China) after staining for annexin V and propidium iodide (21172 Apoptosis Detection Kit, Lumiprobe RUS Ltd., Moscow, Russia).

Techniques: Electron Microscopy