Journal: bioRxiv

Article Title: Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

doi: 10.1101/800011

Figure Lengend Snippet: a The abundance of 97 PTEN variants overexpressed in yeast was assayed by western blot. Quantified values by band densitometry were normalized to WT-PTEN = 1 and empty vector = 0. b Representative scatter plot of single-cell fluorescence intensities from flow cytometry for overexpressed sfGFP-fusions of PTEN WT, D326N and H93Q versus their level of transfection as visualized with mTagRFP-t. c Histogram showing the relative frequency of sfGFP/mTagRFP-t ratio for the same variants in ( b ). d Protein stability of 105 PTEN variants assayed in HEK293 cells by flow cytometry calculated as median of sfGFP/mTagRFPt and expressed as normalized to WT = 1. Data in a and d are expressed as mean of well replicates (n > 4) ± SEM. * indicates p < 0.05 compared to WT by student’s t-test. e Plot of yeast abundance and HEK293 PTEN stability variant data sets. (Pearson r = 0.55, p< 0.0001).

Article Snippet: Cells were permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (FC005, R&D Systems) and stained with 1:100 of Rabbit anti-pAKT (S473, #9271, Cell Signaling) and 1:100 of Mouse anti-pan-AKT (#2920, Cell Signaling) for one hour on ice.

Techniques: Western Blot, Plasmid Preparation, Fluorescence, Flow Cytometry, Transfection, Variant Assay

Journal: bioRxiv

Article Title: Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction

doi: 10.1101/800011

Figure Lengend Snippet: a, b Flow cytometry single-cell rolling median of pAKT/AKT ratios versus expression levels of sfGFP-PTEN in HEK293 cells for WT, C124S and 4A variants and the corresponding frequency histogram in b). Vertical bars indicate median values. c Functional impact of 105 PTEN MS variants on pAKT/AKT levels. Data plotted are median differences between transfected and in-well untransfected cells for each variant Data are expressed as mean of well replicates (n >=4) ±SEM. Values are normalized to WT=1 and no difference to untransfected=0. * indicates p<0.05 compared to WT, # indicates p<0.05 compared to 0 by one-sample Student’s t-test. d Relative pAKT/AKT changes are shown for variants exhibiting dominant negative effects on pAKT/AKT, as well as the constitutively active 4A in both parental and a PTEN-KO HEK293 cell line. e , f Plots of PTEN variant function by pAKT/AKT changes in HEK293 cells versus variant function of genetic interaction with Δ Vac14 in yeast (Pearson r = 0.79, p< 0.0001), and rate of eclosion in Drosophila (Pearson r = 0.78, p< 0.0001).

Article Snippet: Cells were permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (FC005, R&D Systems) and stained with 1:100 of Rabbit anti-pAKT (S473, #9271, Cell Signaling) and 1:100 of Mouse anti-pan-AKT (#2920, Cell Signaling) for one hour on ice.

Techniques: Flow Cytometry, Expressing, Functional Assay, Transfection, Variant Assay, Dominant Negative Mutation