Journal: BioMed Research International

Article Title: RhoA Controls Wnt Upregulation on Microstructured Titanium Surfaces

doi: 10.1155/2014/401859

Figure Lengend Snippet: (a) Total RhoA activation levels in MC3T3 cells growing on Polished or SLA titanium surfaces by G-LISA assay. * P < 0.05 versus Polished. (b) Canonical WNT activity by Luciferase reporter assay in C2C12 cells on Polished or SLA surfaces after RhoA inhibition by dominant negative isoform (dnRhoA) or activation by transfection with constitutively active isoform (caRhoA). * P < 0.01 versus vehicle-treated Polished; # P < 0.01 versus vehicle-treated SLA; § P < 0.01 versus vehicle-treated Polished.

Article Snippet: The dominant negative RhoA (plasmid 12152: pTriEx-RhoA FLARE.sc Biosensor T19N) and constitutively active (plasmid 12151: pTriEx-RhoA FLARE.sc Biosensor Q63L) isoforms were obtained from public Addgene plasmid repository and were kindly shared by the Klaus Hahn Lab [ ].

Techniques: Activation Assay, Activity Assay, Luciferase, Reporter Assay, Inhibition, Dominant Negative Mutation, Transfection

Journal: Animal Cells and Systems

Article Title: ECM stiffness regulates calcium influx into mitochondria via tubulin and VDAC1 activity

doi: 10.1080/19768354.2024.2393811

Figure Lengend Snippet: Impact of ECM stiffness on mitochondrial calcium influx. (A, C) Live imaging time-lapse series capturing the intensity of calcium-specific biosensors within HeLa cells: pCMV CEPIA2 mt for mitochondrial calcium (green) and pCMV R-CEPIA1er for endoplasmic reticulum (ER) calcium (red), post-treatment with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Ionomycin was administered 5 min after initiating the imaging process, with only the media and cells present in the confocal dish during the initial 5 min. The accompanying color scale bar represents calcium levels, with red indicating high and blue denoting low calcium concentrations (scale bar = 10 µm). (B, D) Quantitative analysis of the time courses of normalized emission intensity from the pCMV CEPIA2 mt and pCMV R-CEPIA1er biosensors in HeLa cells treated with 1 µM ionomycin on substrates with 1 and 40 kPa stiffness. Each line represents the mean value of normalized emission intensity for a group of 10 cells (n = 10), and the error bars reflect the standard error of the mean (SEM), illustrating the dynamic calcium changes in response to ECM stiffness and ionomycin treatment.

Article Snippet: To determine the stiffness differences between the 1 and 40 kPa substrates, we initially employed Cyto-FAK (Addgene #78300) to observe focal adhesion kinase (FAK) activity across the two stiffness conditions (Supplementary Figure S1), as described by Seong et al. ( ).

Techniques: Imaging

Journal: Sensors (Basel, Switzerland)

Article Title: Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

doi: 10.3390/s19153253

Figure Lengend Snippet: Diagram of ADP sensor libraries. ( a ) Different relative orientations between the mTFP1 and mVenus FRET pair was achieved through the circular permutation kit developed by Pertz and co-workers ; ( b ) In addition, different linker lengths and linker compositions were screened.

Article Snippet: The genes for the mTFP1 and mVenus wildtype and circularly permuted variants were subcloned from the Pertz Lab cpFRET biosensors kit (Addgene kit # 1000000021) [ ].

Techniques:

Journal: Bio-protocol

Article Title: Sensitive and Adaptable Turn-On Maturation (ATOM) Fluorescent Biosensors for Detecting Subcellular Localization of Protein Targets in Cells

doi: 10.21769/BioProtoc.5239

Figure Lengend Snippet: HeLa cells were transfected with one plasmid expressing ER- or mitochondria-localized y-ATOM SH2 and a second plasmid encoding either ER- or mitochondrial-targeted SH2 (transfection #1), or SH2 with no localization tags (resulting in cytoplasmic SH2 expression; transfection #2). (A) Cells appeared green when the biosensor and ligand were targeted to the same compartment and were mostly dark when the biosensor was targeted to ER or mitochondria and the ligand was expressed in the cytoplasm. Scale bars are 100 μm. For the quantification at the right side, each dot represents one cell (n ≥ 50), and error bars correspond to the standard deviation. (B) Confocal images of HeLa cells expressing y-ATOM SH2 and SH2 ligand, both targeted to ER (left) or mitochondria (right), reveal the expected mesh-like and tubular structures of the respective organelles. Scale bars are 10 μm.

Article Snippet: HeLa human epithelial cells (ATCC, catalog number: CCL-2) 4. pCMV-MBP-yATOM_SH2 for expressing y-ATOM SH2 biosensor in mammalian cells (Addgene plasmid # 209708; http://n2t.net/addgene:209708 ; RRID: Addgene_209708) 5. pCMV-MBP-yATOM_WDR5 for expressing y-ATOM WDR5 biosensor in mammalian cells (Addgene plasmid # 209706; http://n2t.net/addgene:209706 ; RRID: Addgene_209706) 6. pCMV-MBP-yATOM_mCherry for expressing y-ATOM mCh biosensor in mammalian cells (Addgene plasmid # 209707; http://n2t.net/addgene:209707 ; RRID: Addgene_209707) 7. pCMV-MBP-yATOM_RAS for expressing y-ATOM RAS biosensor in mammalian cells (Addgene plasmid # 209709; http://n2t.net/addgene:209709 ; RRID: Addgene_209709) 8. pCMV-RBP_WDR5 for expressing WDR5 in mammalian cells (Addgene plasmid # 209702; http://n2t.net/addgene:209702 ; RRID: Addgene_209702) 9. pCMV-mCherry (Y70A) for expressing the Y70A (nonfluorescent) mutant of mCherry in mammalian cells (Addgene plasmid # 209703; http://n2t.net/addgene:209703 ; RRID: Addgene_209703) 10. pCMV-SH2 for expressing SH2 domain in mammalian cells (Addgene plasmid # 209704; http://n2t.net/addgene:209704 ; RRID: Addgene_209704) 11.

Techniques: Transfection, Plasmid Preparation, Expressing, Standard Deviation