Journal: Cancer research communications

Article Title: A miniaturized screening platform to identify novel regulators of extracellular matrix alignment

doi: 10.1158/2767-9764.CRC-22-0157

Figure Lengend Snippet: Fibroblasts were plated on Day 0 in gelatin-coated 384-well plates. The media was changed on Day 1 and 3 with the addition of ascorbic acid, fluorescently labeled fibronectin, and experimental treatments. On Day 5, the samples were fixed, and the nuclei stained. Automated confocal imaging was used to acquire images of the samples in the plate, with 8 μm z-stacks taken at 4 positions within each well.

Article Snippet: Briefly, 10 mg of bovine fibronectin (MilliporeSigma, Cat. #F4759) was suspended in PBS at 1 mg/mL.

Techniques: Labeling, Staining, Imaging

Journal: Cancer research communications

Article Title: A miniaturized screening platform to identify novel regulators of extracellular matrix alignment

doi: 10.1158/2767-9764.CRC-22-0157

Figure Lengend Snippet: Image analysis was automated using a MATLAB script. ImageJ/FIJI was run under the control of MATLAB using the Miji plugin. Images were imported to MATLAB, passed to ImageJ, and Z-projections of the nuclei channel were thresholded, made binary, segmented, and counted. Z-projections of the fibronectin channel were analyzed using OrientationJ, which outputs a histogram of fiber orientation. This output is passed back to MATLAB and the histogram is centered at the mode. The fraction of fibers within 20° of the mode was determined as a metric of matrix alignment.

Article Snippet: Briefly, 10 mg of bovine fibronectin (MilliporeSigma, Cat. #F4759) was suspended in PBS at 1 mg/mL.

Techniques: Control

Journal: Cancer research communications

Article Title: A miniaturized screening platform to identify novel regulators of extracellular matrix alignment

doi: 10.1158/2767-9764.CRC-22-0157

Figure Lengend Snippet: A. Images from this study depicting examples of sufficient fibrillogenesis, poor fibrillogenesis, and imaging error. White arrow indicates a hole in the matrix, which signifies poor fibrillogenesis. B. Histogram of percentage of pixels below background for every field of view (FOV) imaged in this study (n=4336). C. To ensure sufficient fibrillogenesis and imaging quality for alignment analysis, the fraction of pixels in the fibronectin channel that fall below background noise levels (100 a.u) is quantified for each FOV. Those with >10% of pixels below background were flagged and excluded from subsequent analysis. Drug screen wells containing less than 3 valid FOV were excluded from subsequent analysis, and drugs with 0 or 1 valid wells across 3 replicate plates were also excluded. Images in Panel C show FDM treated with 1μM of PP-242 (hit #13 in kinase inhibitor screen). D. Out of 924 total wells analyzed in this study, 152 were excluded for insufficient matrix, 4 were flagged for high toxicity, and another 77 were flagged for both conditions.

Article Snippet: Briefly, 10 mg of bovine fibronectin (MilliporeSigma, Cat. #F4759) was suspended in PBS at 1 mg/mL.

Techniques: Imaging

Journal: eLife

Article Title: Burst mitofusin activation reverses neuromuscular dysfunction in murine CMT2A

doi: 10.7554/eLife.61119

Figure Lengend Snippet:

Article Snippet: On day 5 cells were re-plated on poly-ornithine/laminin/fibronectin (Sigma, Cat# P4957, # L2020, # F4759) coated 18 mm glass coverslips and on the following day changed to neuronal medium supplemented with Dox (1 µg/ml added every other day), valproic acid (1 mM; Sigma, Cat# 676380), dibutyryl cAMP (200 µM; Sigma, Cat# D0627), BDNF, NT-3, CNTF, GDNF (all 10 ng/ml, Peprotech, Cat# 450–02, #450–03, #450–13, #450–10), retinoic Acid (1 µM; Sigma, Cat# R2625) and antibiotics.

Techniques: Knock-Out, Transfection, Construct, Plasmid Preparation, Recombinant, Sequencing, Mutagenesis, Electron Microscopy, Software