matlab-based cellprofiler pipeline  (MathWorks Inc)

Journal: Computational and Structural Biotechnology Journal

Article Title: Spatial patterns of the cap-binding complex eIF4F in human melanoma cells

doi: 10.1016/j.csbj.2023.01.040

Figure Lengend Snippet: In situ detection of eIF4F subcellular distribution. (A) Left panel: schematic view of the proximity ligation assay for the eIF4E-eIF4G complex. 4E: eIF4E, 4 G: eIF4G, 4 A: eIF4A, AAA: polyA tail. Right panel: example images of the eIF4E-eIF4G PLA assay and cell segmentation. The eIF4E-eIF4G complex was stained following the proximity ligation assay protocol, the cytoskeleton was stained by Phalloidin-Alexa 488, and the nucleus was stained with Hoechst 33342. (B) Polysome profile and eIF4E-eIF4G PLA assay of A375 cells upon treatment with PP242. Cells were treated with PP424 at 1 μM for 24 h, and then lysed by polysome hypotonic buffer or fixed with 4% PFA. (C) Polysome profile and eIF4E-eIF4G PLA assay of QBC989 cholangiocarcinoma cells upon nutrient deprivation. Cells were cultured with HBSS solution for nutrient starvation for 16 h, followed by hypotonic buffer lysis or 4% PFA fixation. (D-E) A375 cells expressing the ERK-KTR reporter gene were treated with 1 μM vemurafenib for 24 h and the eIF4E-eIF4G PLA assay was performed. Single cell quantification of ERK-KTR nuclear translocation (p-ERK1/2) and eIF4E-eIF4G spot count are plotted. (F) Pipeline of the eIF4E-eIF4G PLA image analysis. The eIF4E-eIF4G complex was stained following the proximity ligation assay protocol, the cytoskeleton was stained with Phalloidin-Alexa 488, and the nucleus was stained with Hoechst 33342. Cell images were then subjected to Cellprofiler 2.0 analysis to identify the nucleus and cytoplasm. The eIF4E-eIF4G spots were identified by using Cellprofiler module ‘IdentifySpots.m′ followed by correlation with each cell. The localization pattern of the eIF4E-eIF4G spots was calculated with Cellprofiler module ‘MeasureLocalizationOfSpots.m′. (G) Example images of the cell segmentation and spot localization analysis. (H) Summary of the spot features and cell features measured by Cellprofiler 2.0.

Article Snippet: For the MATLAB-based Cellprofiler pipeline, we used the Cellprofiler modules in combination with the modules developed by Lucas Pelkmans’s lab . We shared the ‘MeasureLocalizationOfSpots.m′ file in the supplemental files, however, please refer to the work from Battich et al. Battich et al., when using this code.

Techniques: In Situ, Proximity Ligation Assay, Staining, Cell Culture, Lysis, Expressing, Translocation Assay