Fig 1: GDPD5 expression affects cell surface levels of certain GPI‐AP in endothelial cells. A) HUVEC were transfected with either mCerulean‐C1 empty vector (control), mCerulean‐GDPD5 or mCerulean‐GDPD5‐H233A, respectively, and subjected to a flow cytometry assay 24 h post transfection. Therefore, cells were detached, fixed and stained for the surface GPI‐AP signal using FLAER‐AF488. For each sample, 10000 mCerulean expressing cells were counted and analyzed for their AF488 fluorescence intensity. Histograms show FLAER‐AF488 intensity distributions with the right shifted curve indicating increased surface GPI‐AP (left). Quantification of the relative surface levels of GPI‐AP analyzed via flow cytometry of the different transfected cell populations is shown on the right (quantification based on median fluorescence intensity values detected above control antibody threshold). Error bars = SEM, n = 8 experiments. Significance was tested using repeated measures ANOVA with Dunnetts test for multiple comparisons (** p <0.01, **** p <0.0001, ns = not significant). B) HUVEC were co‐transfected with either mCerulean‐C1 empty vector (control), mCerulean‐GDPD5 or mCerulean‐GDPD5‐H233A and the respective FLAG‐tagged GPI‐AP target (CD59‐FLAG, NT5E‐FLAG or TFPI‐FLAG). 24 h post transfection, cells were detached, fixed and stained for the surface FLAG signal using anti‐FLAG(M1)‐DL488 antibodies. For each sample, 10000 mCerulean expressing cells were counted and analyzed for their DL488 fluorescence intensity. Histograms show the FLAG‐DL488 intensity distribution with a right shift indicating more surface localized GPI‐AP. C) Quantification of the data shown in A. Error bars = SEM, n ≥ 7 experiments. Significance was tested using ordinary one‐way ANOVA with Dunnetts test for multiple comparisons. Paired Wilcoxon test or paired t‐test were used when the number of experiments wasn't the same for all samples. (** p <0.01, **** p <0.0001, ns = not significant).
Fig 2: CD59 and TFPI are targets of GDPD5 on the endothelial cell surface. A) HUVEC were transfected with 200 pmol of siCtrl or siGDPD5, kept for 48 h, and transfected again with the same amounts of the respective siRNAs. 24 h after the second transfection, HUVEC were detached, fixed and stained for the surface CD59 level using anti‐CD59‐FITC antibodies. For each sample, 20000 cells were counted and analyzed for their FITC fluorescence intensity. B) HUVEC were transfected with 200 pmol of siCtrl or siGDPD5, kept for 48 h, and transfected again with the same amounts of the respective siRNAs and the GPI‐AP target indicated. 24 h after the second transfection, HUVEC were detached, fixed and stained for the surface FLAG signal using anti‐FLAG(M1)‐DL488 antibodies. For each sample, 20000 cells were counted and analyzed for their DL488 fluorescence intensity. C) Quantification of the data shown in B (carried out as described in legend to Figure 5). Error bars = SEM; n = 5 for NT5E‐FLAG; n ≥ 8 experiments for CD59‐FLAG or TFPI‐FLAG. Significance tested with paired t‐test or paired Wilcoxon test (** p < 0.01, ns = not significant). D) HUVEC were either transfected with GDPD5‐EGFP or left untransfected and then cultured for 32 h. Cells were washed and subsequently incubated with starvation medium for 1 h. Medium was collected (untreated samples) and cells were incubated with starvation medium supplemented with 500 µM histamine for 1 h. Medium was again collected (stimulated samples) and samples were analyzed for their soluble CD59 content via CD59‐ELISA. n = 10, significance tested with paired Wilcoxon test (left) or paired t‐test (middle, right) (** p < 0.01, *** p < 0.001).
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