Fig 2: Identification of reproducible broad-spectrum phosphatase and tensin homolog-synthetic sick/lethal (PTEN-SSL) genes. a Cmparison of the secondary short hairpin RNA (shRNA) screen described in this study and three independent RNA interference (RNAi) screening datasets selected for reanalysis: Breast Functional Genomics [17], Cancer Dependency Map [19], and Kinase Dependency Profiles [18]. b The p values from the one-sided Wilcoxon rank sum test for whether PTEN- cell lines were more sensitive (lower ATARiS scores) to knockdown of each gene compared to PTEN+ cell lines. Each bar represents one of 727 genes in the secondary screen that had ATARiS gene solutions. Possible broad-spectrum PTEN-SSL hits were selected using the same criterion (p < 0.05, uncorrected for multiple testing) for each screen and are shown as red bars. PIK3CB, NUAK1, STK11, ADAMTS20, AP1M2, and HMMR were found to be hits in our reanalysis of = 1 of the previous screens. c Number of overlaps in possible broad-spectrum PTEN-SSL genes that were identified by this study and in reanalysis of three previous screens; p values based on one-sided hypergeometric distribution

Fig 3: HOIP associates with PTEN and modulates PTEN stability. A. Intracellular localization analysis of HOIP and PTEN by immunofluorescence assay. AGS cells were cultured in normal medium before fixation. Intracellular localization of PTEN (red) and HOIP (green) were shown. Nuclei (blue) were stained with 40,6-diamidino-2-phenylindole (DAPI). B and C. Co-IP assay reveals association between endogenous HOIP and PTEN in AGS cells. AGS cells were harvested with RIPA lysis buffer. CO-IP was performed using antibody as indicated. D. In the presence of the proteasome inhibitor MG132, HOIP could not further increase PTEN protein levels. AGS cells were transfected with 50 μM siControl or siHOIP. After 24 h, cells were treated with 10 µM MG132/vehicle for 6h. Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. E and F. HOIP depletion increases PTEN half-life in AGS cells. AGS cells were transfected with 50nM siControl or siHOIP. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot analysis. The results are representative for three independent experiments. The PTEN relative density was measured by Image J software.

Fig 4: Changes provoked by aerobic exercise training on microRNA-1 downstream-associated pathways in patients with chronic heart failure with reduced ejection fraction (HFrEF). Note that despite the increase in microRNA-1 levels, the PI3K-AKT pathway is increased, which seems to be associated with a reduction in PTEN levels. In addition, HDAC4 levels decrease, which leads to an increase in follistatin, MEF2C, and MyoD levels. These changes improve protein synthesis, skeletal muscle differentiation, and myocyte fusion. The consequence of such changes is an increase in muscle cross-sectional area.

Fig 5: TUSC3 associates with oligosaccharyltransferase subunit STT3B.(a) TUSC3 co-immunoprecipitates with STT3B in HEK293T cells. Lysates from HEK293T cells were immunoprecipitated with TUSC3 antibody or control IgG and detected with STT3B antibody on a western blot. IgG heavy chain (IgG HC) represents the loading control. Endogenous input STT3B and TUSC3 levels are shown. (b) HEK293T cells were transfected with TUSC3 or an empty control pcDNA 3.1 vector (V). Lysates from transfected cells were immunoprecipitated using STT3B antibody or control IgG and TUSC3 was detected using western blot. IgG light chain (IgG LC) serves as loading control. TUSC3 overexpression affects ER-Luciferase N-glycosylation in HEK293T cells. (c) HEK293T cells were transfected with wild type firefly luciferase (Luc) or luciferase fused to an endoplasmic reticulum targeting sequence (ER-Luc) and seeded in 96-well plates. Luminesence was measured 32–48 hours after transfection and normalized to the cell count. (d) HEK293T cells were co-transfected with ER-Luc and empty pcDNA 3.1 vector (V) or full length TUSC3. Luminesence was measured and normalized to the cell count. The luciferase experiments are representative of several biological replicates and were performed in triplicates (depicted as means and standard deviations). Electrophoretic gel shift of the fusion construct (ER-Luc) in contrast to wild type luciferase (Luc) is shown. TUSC3 overexpression does not have an effect on the molecular weight of the ER-Luciferase. (e) A panel of cell lines derived from human prostate cancers (DU145, PC3, LNCaP) and benign prostatic hyperplasia (BPH-1) were assessed for expression of TUSC3, PTEN and ENTPD5. Cropped blots are depicted for clarity reasons and full-length blots will be provided upon request.