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Dual knockdown of MK-STYX and PTPMT1 is sufficient to resensitize MK-STYX knockdown cells to chemotherapeutic treatment.

<p>(A) HeLa cells were plated on an electrode-containing plate and viability was measured using the xCELLigence system. Cells were transfected for 30 hours and incubated in the presence of 50 nM paclitaxel or a vehicle-only control for an additional 24 hours. Mean cell viability values following both drug (dark gray bars) and vehicle (light gray bars) treatments are shown. Bars represent standard deviation of at least triplicate measurements. Unpaired, two-tailed t-tests: **p≤0.01; ***p≤0.001. (B–E) HeLa cells were transfected with control (B–D, gray circles), MK-STYX (B, blue circles), PTPMT1 (C, green circles), or MK-STYX+PTPMT1 siRNAs (D, red circles) for 30 hours before being treated with a dose response of paclitaxel for an additional 24 hours. Mean EC₅₀ values were determined for each condition using GraphPad PRISM (plotted in E; indicated on each plot). Bars represent standard deviation of four measurements per condition. Unpaired, two-tailed t-tests: ***p≤0.001.</p

MK-STYX knockdown fails to protect cells from the loss of viability caused by PTPMT1 depletion.

<p>(<b>A–B</b>) HeLa cells were transfected with a pool of two unique MK-STYX siRNAs (A), a pool of two unique PTPMT1 siRNAs (B), or a non-targeting control siRNA (A and B) for 30 hours. Relative knockdown was assessed via RT-PCR. Values represent mean mRNA levels normalized to control siRNA-treated cells using HPRT1 as a reference gene. Error bars represent standard deviation. (<b>C</b>) HeLa cells were transfected with control, MK-STYX, PTPMT1, or MK-STYX + PTPMT1 siRNAs for 24 hours before cellular viability was assayed in real-time using a xCELLigence plate reader.</p

MK-STYX interacts with PTPMT1.

<p>(A) TAP-tagged MK-STYX was expressed and immunopurified from HeLa cells. Endogenous interaction partners were identified by LC-MS/MS and were bioinformatically filtered against a large control dataset (∼350 non-MK-STYX TAP tag experiments) to identify unique and significant interactions with MK-STYX. The gene name of each protein identified is listed in order of significance (the most significant hits listed at the top). A p value (demonstrating the significance of each interaction partner), and interactor score (calculated based on the p value, number of replicates in which the interactor was identified, uniqueness of the MK-STYX interaction relative to the control interactions, and MASCOT scores and total coverage of the peptides) is shown for each interactor (lower p-value and/or higher interactor score correlates with a stronger molecular interaction with MK-STYX). (<b>B</b>) The mitochondrial localization of top MK-STYX interaction partners is shown (gray rectangle: bait; white circle: interaction partner; line: interaction). (<b>C</b>) V5-MK-STYX and FLAG-PTPMT1 (wildtype) or FLAG-PTPMT1 C132S (a catalytically inactive mutant) were transfected into 293FT cells and FLAG immunoprecipitated. Input lysates or immunoprecipitates were immunoblotted with the indicated antibodies. The * indicates light chain of the antibody used to immunoprecipitate the protein.</p