Journal: Cellular signalling

Article Title: Phospho-PTM Proteomic Discovery of Novel EPO- Modulated Kinases and Phosphatases, Including PTPN18 as a Positive Regulator of EPOR/JAK2 Signaling

doi: 10.1016/j.cellsig.2020.109554

Figure Lengend Snippet: (A) Wild-type PTPN18 and PTPN18-Y389F constructs, and matched ectopic expression levels in stably transduced UT7epo cells: UT7epo cells were stably transduced with lentiviruses encoding wild-type PTPN18, PTPN18-Y389F or an empty vector negative control construct (EVEC). Levels of ectopic expression (“EE”) of PTPN18 and PTPN18-Y389F constructs in derived cell lines were assessed by western blotting (lower sub-panel). (B,C) Ectopically expressed PTPN18 promotes UT7epo cell proliferation and survival with dependency on EPO- dose, and PTPN18-Y389F: In cell growth assays, exponentially growing UT7epo-EE-PTPN18, EE-PTPN18-Y389F and EE-EVEC cells (each at 5 to 7×105 cells/mL) were washed and plated (at 1×105 cells/mL) in EPO at 0.15, 0.3, 0.6, or 1.2 U/mL. At day 3.5, viable cell numbers (B), and frequencies of non-viable cells were determined (C). In B and C, graphed values are means +/− SE (n=3). (D) PTPN18, and PTPN18-Y389F effects on UT7epo cell growth are persistent: UT7epo- EE-PTPN18, EE-PTPN18-Y389F and EE-EVEC cells were plated as in panel B. At 4-day intervals, cultures were passed volumetrically to fresh medium with EPO at 0.3 U/mL. At day-21, numbers of viable cells and frequencies of non-viable cells were determined (mean values +/− SE, n=3) (left panel). For comparison, data for 3-d assays for cells cultured in EPO at 0.6 U/mL are provided (mean values +/− SE, n=3). (E) PTPN18 effects on UT7epo cell survival following EPO withdrawal: Exponentially growing UT7epo- EE-PTPN18, EE-PTPN18Y389F, and EE-EVEC cells were washed thrice and plated in IMDM, 10% FBS in the absence of EPO. Viability then was assayed at 20h and 24h post plating. At 20h, viability for each cell population was ≥95% (data not shown). At 24h, and as assayed via Annexin-V staining, significant cytoprotective effects were exerted by ectopically expressed PTPN18 (EE-PTPN18), and to a lesser extent EE-PTPN18-Y389F. Graphed values are means +/− SE (n=3). Representative primary flow cytometry data are also illustrated (right sub-panel).

Article Snippet: In phospho-tyrosine PTM proteomic analyses, UT7epo cells were plated at 1×10 5 cells/mL in IMDM (HyClone, #SH30228.01), 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin sulfate (“P/S”), with rhEPO at 2.5 U/mL (Procrit®, Amgen).

Techniques: Construct, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Negative Control, Derivative Assay, Western Blot, Cell Culture, Staining, Flow Cytometry

Journal: Cellular signalling

Article Title: Phospho-PTM Proteomic Discovery of Novel EPO- Modulated Kinases and Phosphatases, Including PTPN18 as a Positive Regulator of EPOR/JAK2 Signaling

doi: 10.1016/j.cellsig.2020.109554

Figure Lengend Snippet: UT7epo cells stably transduced with wt-PTPN18 (“EE-PTPN18”), PTPN18-Y389F (“EE-PTPN18-Y389F”) or empty vector (EVEC) lentiviruses were plated at 1×105 cells/mL, and expanded to exponential growth phase (5 to 7×105 cells/mL). Cells were washed and plated at 7×105 cells per mL in IMDM 10% FBS without EPO. At 20 hours post EPO withdrawal, cells were challenged with EPO (1.5 U/mL), and at the indicated time points (0, 5, 15, 45 minutes) cells were brought to 2˚C, washed and processed for total protein extracts. Via western blot analyses, levels of activation site phosphorylated, and total ERK1/2, AKT, STAT5 and JAK2 were then determined. (A) PTPN18 sustains the EPO- induced activation of ERK1/2, and AKT with Y389- dependency: Cell lysates from UT7epo- EE-PTPN18, EE-PTPN18Y389F and EE-EVEC cells were analyzed by western blotting. Results shown are representative of two independent experiments. (B) PTPN18 sustains the EPO- induced activation of STAT5 and JAK2 with Y389- dependency: Cells, lysates and samples assayed for phospho- and total STAT5, and phospho- and total JAK2 are those analyzed in panel A. Results shown are representative of two independent experiments. (C) Summarized effects of PTPN18 on EPO- induced ERK1/2, AKT and JAK2/STAT5 activation, and the heightened proliferation and survival of erythroid progenitor UT7epo cells: The model outlined illustrates the EPOR, JAK2 and the EPOR associated adaptor protein RHEX together with PTPN18’s observed positive effects on ERK1/2, AKT and JAK2/STAT5 signaling modules.

Article Snippet: In phospho-tyrosine PTM proteomic analyses, UT7epo cells were plated at 1×10 5 cells/mL in IMDM (HyClone, #SH30228.01), 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin sulfate (“P/S”), with rhEPO at 2.5 U/mL (Procrit®, Amgen).

Techniques: Stable Transfection, Transduction, Plasmid Preparation, Western Blot, Activation Assay