Journal: PLoS Genetics

Article Title: Revertant Mutation Releases Confined Lethal Mutation, Opening Pandora's Box: A Novel Genetic Pathogenesis

doi: 10.1371/journal.pgen.1004276

Figure Lengend Snippet: (A) Gap junction formation by the transfected Cx26 variants. Each panel contains two co-transfected cells connected to each other. Wild-type, p.Gly45Glu/p.Tyr136X and p.Gly45Glu mutants of Cx26 were tagged with monomeric Red Fluorescent Protein (mRFP) and co-transfected with Green Fluorescent Protein (EGFP)-tagged Cx26 p.Val27Ile/p.Glu114Gly into HeLa cells as indicated. Gap junction formation sites are indicated by arrowheads. The combination of WT Cx26 and Cx26 p.Val27Ile/p.Glu114Gly (top row) results in gap junctions that consist of both Cx26 proteins (yellow signal). The combination of Cx26 p.Gly45Glu/p.Tyr136X and Cx26 p.Val27Ile/p.Glu114Gly (middle row) results in gap junctions with only Cx26 p.Val27Ile/p.Glu114Gly (green signal). No apparent gap junction formation is seen when Cx26 p.Gly45Glu and Cx26 p.Val27Ile/p.Glu114Gly are cotransfected (bottom row). (B) Aberrant gate opening detected with neurobiotin uptake assay. Fluorescent-tagged Cx26s were cotransfected into HeLa cells as indicated, and treated with neurobiotin in a calcium-free condition. Uptake was detected with AlexaFluor350 streptoavidin dye (blue). Aberrant uptake of neurobiotin is observed only in cells cotransfected with Cx26 p.Gly45Glu and Cx26 p.Val27Ile/p.Glu114Gly (bottom row).

Article Snippet: Briefly, cells were washed with calcium free Hank's buffered salt solution for 20 minutes and incubated with phosphate-buffered saline (PBS) containing 0.1 mg/ml neurobiotin (Vector Laboratories) for another 20 minutes.

Techniques: Transfection

Journal: Matrix Biology Plus

Article Title: Profiling of collagen and extracellular matrix deposition from cell culture using in vitro ExtraCellular matrix mass spectrometry imaging (ivECM-MSI)

doi: 10.1016/j.mbplus.2024.100161

Figure Lengend Snippet: Workflow for in Vitro ExtraCellular Matrix Mass Spectrometry Imaging (ivECM-MSI). 1.) Cells are seeded on Nunc Lab-Tek II glass chamber slides. Gelatin can be used as a coating prior to seeding if certain cell lines are not adhering to the glass. 2.) Cells are incubated in the glass chambers slides until confluence. 3.) The medium is removed and each well in the chamber slide is then decellularized with a 20 mM ammonium hydroxide solution for 5 min. Each well is then rinsed 4 times with sterile water. Next, the chambers are removed from the slide. Afterword, the slide is dried in a vacuum desiccator. Slides can be stored in a −20 °C freezer for temporary storage if needed. 4.) A 0.1 mg/mL solution of collagenase III is sprayed evenly onto the slides. The collagenase sprayed slides are then incubated in a 37 °C humidity chamber for 5 h to digest the collagen into peptides. Afterword, the slides are dried in a vacuum desiccator. Slides can be stored in a −20 °C freezer for temporary storage if needed. 5.) The collagenase digested slides are then sprayed evenly with a 7 mg/mL α-cyano-4-hydroxycinnamic acid (CHCA) solution. Immediately after CHCA spraying, a 5 mM ammonium phosphate solution is sprayed on top of the MALDI matrix to minimize matrix cluster formation. Slides at this stage can be temporarily stored in a vacuum desiccator if needed. 6.) The matrix sprayed slides are loaded into the MALDI mass spectrometer for MALDI mass spectrometry analysis. 7.) Finally, the data is visualized through mass spectrometry imaging heatmaps, m / z features are searched through a collagen peptide database, and statistics are performed. Created with BioRender.com .

Article Snippet: A 0.1 mg/mL collagenase III (Worthington, CAT# LSOO4180) solution was made in a 1 mM calcium chloride (Sigma Aldrich, C1016) and 10 mM ammonium bicarbonate (Sigma Aldrich, A6141) buffer.

Techniques: In Vitro, Mass Spectrometry, Imaging, Incubation, Sterility