sted abberior stedycon instrument  (abberior instruments)

Journal: Frontiers in Oncology

Article Title: Focal adhesion kinase promotes ribosome biogenesis to drive advanced thyroid cancer cell growth and survival

doi: 10.3389/fonc.2025.1252544

Figure Lengend Snippet: NPM1 is necessary for pY397 FAK localization to the nucleolus in adherent cells (A) BCPAP cells were probed with antibodies against NPM1, FBL, and UBTF and imaged using STED microscopy. At least 50 cells were visualized in three independent experiments. Scale bar represent 1μm. (B) Percent of co-localization between pY397 FAK and NPM1, UBTF, and FBL respectively using Integrated Density Analysis by Image J. (C) 8505C cells were knocked down with either a non-targeting control or three shRNAs targeting NPM1 prior to immunoblotting with antibodies against NPM1, pY397 FAK, total FAK, or α-tubulin. Quantification of NPM1 expression is normalized to α-tubulin and sh-COO2 control and is listed under the NPM1 blot. MW marker is shown on the left of each blot in kDa. Two independent experiments were performed. (D) Immunofluorescence was performed in 8505C shCOO2 or shNPM1 cells probed with antibodies against pY397 FAK, FBL, and DAPI. Cells were imaged at 60X confocal imaging in three independent experiments with visualization of at least 50 cells. Scale bar represent 5 μm. (E) Percent of co-localization between pY397 FAK and FBL in our non-targeting controls and shNPM1 knockdown cells using Integrated Density Analysis by Image J. Results displayed as mean +/- SEM. *, p<0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Images were acquired on STED Abberior Stedycon Instrument in the ALMC.

Techniques: Microscopy, Control, Western Blot, Expressing, Marker, Immunofluorescence, Imaging, Knockdown

Journal: Frontiers in Oncology

Article Title: Focal adhesion kinase promotes ribosome biogenesis to drive advanced thyroid cancer cell growth and survival

doi: 10.3389/fonc.2025.1252544

Figure Lengend Snippet: NOP56 co-localizes with pY397 FAK and promotes thyroid cancer growth (A) BioID workflow representing FAK fused to a promiscuous biotin ligase and interacting partners of FAK being biotinylated, pulled down, and identified by mass spectrometry. (B) Immunoblotting of BCPAP cells transduced with EV, WT, and Y397F FAK BioID2 and probed with antibodies against pY397 FAK, total FAK, and α-tubulin. The higher MW band represents the 27kDA BioID2 construct fused to WT or Y397 FAK and lower MW band is the endogenous FAK band. (C) STRING analysis of significantly enriched proteins reveals known interacting partners of FAK. Blue connections signify known interactions from database. Red connections signify experimentally determined known interactions. (D) FAK interacting partners were identified by analyzing proteins that bind to WT FAK in both datasets and enriched at least 2-fold change compared to EV. Gene Ontology Pathway Analysis was conducted for molecular function pathways with a false discovery rate (FDR) <0.05. (E) Gene Ontology Pathway Analysis was conducted for cellular compartment pathways with FDR < 0.05. (F) STRING analysis of nucleolar proteins identified by Gene Ontology Pathway Analysis. Blue connections signify known interactions from database. Red connections signify experimentally determined known interactions. (G) Confocal microscopy of KTC2 cells was performed with antibodies against pY397 FAK and NOP56. At least 50 cells were imaged at 100X with the Abberior Stedycon microscope in two independent experiments. Scale bar represent 2 μm. (H) Immunoblotting of KTC2 cells transduced with a non-targeting control or three NOP56 shRNAs was performed with antibodies against NOP56 and α-tubulin. MW marker is shown on the left of each blot in kDa . (I) Clonogenic assays of KTC2 cells transduced with non-targeting control or NOP56 shRNA were performed. Colonies were stained with crystal violet and imaged after 10 days. Integrated Density was utilized to quantify clonogenic growth using LICOR Odyssey software. Experiments were performed in biological triplicate experiments. Results are displayed as mean +/- SEM. *, p < 0.05.

Article Snippet: Images were acquired on STED Abberior Stedycon Instrument in the ALMC.

Techniques: Mass Spectrometry, Western Blot, Transduction, Construct, Confocal Microscopy, Microscopy, Control, Marker, shRNA, Staining, Software