Fig 2: PTEN variant impact on rat neural growth and sensorimotor behavior in C. elegans.a Representative immunofluorescence images of proximal dendrites from primary cultured hippocampal pyramidal neurons co-stained for the postsynaptic markers of excitatory (PSD-95) and inhibitory (Gephyrin) synapses. Similar results were seen in 128 replicates of WT PTEN, 109 replicates for GFP Control, and 75 replicates for C124S. Scale bar = 5 µm. b Representative images of cultured hippocampal and DRG neurons expressing GFP alone, or with WT-PTEN and PTEN-C124S (scale bar = 100 µm for Hippocampus, and 60 µm for DRG). Similar results were seen in replicates for WT PTEN (132 hippocampus, 463 DRG), GFP Control (125 hippocampus, 488 DRG), and C124S (84 hippocampus, 444 DRG). Effect of overexpression of 20 3XHA-tagged PTEN variants on excitatory synapses (c), soma size (d), total dendritic arbor length (e) in hippocampal neurons, and axonal length in DRG neurons (f). Data are expressed as means ± SEM. *p < 0.05 by two-tailed Satterthwaite approximation as in Fig. 1 (Methods section). g Wild type C. elegans (cePTEN) exhibit chemotaxis towards a NaCl source, while worms harboring a NS mutation in the worm homolog daf18 (cePTEN(rf)) exhibit chemotaxis deficits, which is rescued by overexpression of human PTEN (cePTEN(rf)+hPTEN). h Impact of 20 non-tagged PTEN variants on NaCl chemotaxis in C. elegans is shown by scoring salt preference as (A–B)/(A+B). Data are expressed as means ± SEM and are normalized to cePTEN(rf) + hPTEN = 1 and cePTEN(rf) = 0. Variant data represented in all histograms are color-coded by variant category depicted in the color table. *p < 0.05 compared to WT, #p < 0.05 compared to cePTEN(rf) by two-tailed Satterthwaite approximation. Individual variant means, error, n and nominal p-values for all plots are provided in the Source Data file.

Fig 3: PTEN variants destabilize PTEN.a The abundance of 97 PTEN variants overexpressed in yeast was assayed by western blot. Variants are color-coded by category depicted in the color table at the bottom right. Quantified values by band densitometry were normalized to WT-PTEN = 1 and empty vector = 0. Data is expressed as mean ± SEM. *p < 0.05 compared to WT by two-tailed Satterthwaite approximation (Methods section). b Representative scatter plot of single-cell fluorescence intensities from flow cytometry for overexpressed sfGFP- fusions of PTEN WT (light purple), D326N (pink), and H93Q (dark purple) 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 is expressed as mean of well replicates ±SEM, and variants are color-coded by category depicted in the color table at the bottom right. *p < 0.05 compared to WT by two-tailed Student’s t test. e Plot of yeast abundance and HEK293 PTEN stability variant data sets with color coding as in a and d. Data are expressed as means ± SEM, Pearson r = 0.55, p < 0.0001. Individual variant means, error, n and nominal p-values for all plots are provided in the Source Data file.

Fig 4: Functional and stability data identifies distinct mechanisms of molecular dysfunction.a A normalized plot displaying stability values subtracted from function values for each assay with variants displayed according to their amino acid position below a schematic of PTEN structural domains. Normalized function – normalized stability scores are depicted as a heat map in which a score of 0 (white) indicates variants whose function matches their stability. A positive score (orange) indicates higher function than predicted from instability, while negative scores (magenta) indicates greater dysfunctional than instability (color scale at top right). b Variant function-stability scores for pAKT/AKT assay in HEK293 cells plotted against amino acid position with variants separated into N-terminus, WPD-loop and P-loop domains (red), and variants outside these domains (blue). Data are expressed as means ± SEM. c Frequency distribution of normalized function - stability scores for pAKT/AKT assay in HEK293 assay showing two distinct populations, with red and blue bars denote variants as in b. ***p < 0.0001 by two-tailed Student’s t test. d HEK293 pAKT/AKT assay data plotting PTEN variant function vs. stability with data separated by variants as in b: functional dysfunction of variants that is explained by stability (blue), and not (red). e High correlations are found for variant impact on multiple assays for variants in which dysfunction is associated with instability (blue variants in b–d). Color scale at right. f Weaker cross-assay correlations are seen for variants in catalytic domains, which exhibit greater dysfunction then explained by instability (red variants in b–d). Color scale at right. Individual variant means, error, n and nominal p-values for plot are provided in the Source Data file.

Fig 5: Severity of variant dysfunction correlate with occurrence in cancer.Distribution of normalized PTEN variant function by its frequency within the COSMIC database, with variants with a frequency of 0 in purple, 1–7 reports in green, and 8 or more reports in tan. Mean PTEN function is plotted as bars ±SEM. *p < 0.05, **p < 0.005, ***p < 0.0005 by two-way ANOVA. Individual variant means, error, n and nominal p-values for plot are provided in the Source Data file.