Figure S1. The platelet purity, gating... | Rockefeller University Press

Figure S1.

$The platelet purity, gating strategy, and human NSCLC platelet assays. (A) Representative contour or pseudocolor plots for gating strategy (left) and platelet purity (CD41+) are shown for FC assays performed on washed platelets. (B) The FC histogram overlay showing washed platelets stained with anti–TLT-1 (solid blue) or its isotype control (unfilled blue) and the unstained control sample in solid gray. (C) The quantitation (mean ± SEM) of TLT-1–relative mRNA normalized to β-tubulin in individual samples. Statistical significance tested by Mann–Whitney test. (D) Quantitation of total TLT-1 platelet expression by Western blot. Statistical significance tested by Mann–Whitney test. (E) The FC histograms overlay for positive control for PAC1 marker where washed healthy platelets were stained for PAC1 after activation with ADP (green) or without any external stimulation (blue). The signal from unstained platelets is shown in gray. The events (30,000) were acquired at low flow rate and data analyzed by FlowJo. (F and G) Representative FC pseudocolor plots of washed platelets from NSCLC patients and controls show the NAO-stained platelets where NAO-ve population represents ex vivo apoptotic platelets with their quantitation (G; n = 15 for control and n = 24 for NSCLC). The assay measures the loss of mitochondrial integrity by measuring NAO fluorescence loss from platelets. Statistical significance tested by Mann–Whitney test. (H and I) Representative Western blot–based analysis (H) of total platelet expression of activated Caspase 3, Bax, and Bcl2 proteins in NSCLC and control and their quantitation (I) normalized to loading control. The band densities were measured by ImageJ software and normalized to β-tubulin (loading control). (J) Quantitation of PMV expressing TLT-1 as measured by FC. Statistical significance tested by Mann–Whitney test. (K) ELISA-based comparison of quantitation of TLT-1 in isolated microvesicle fraction versus soluble fraction representing microvesicle-depleted plasma. Each bar represents one patient or control sample as indicated. The data in this figure are presented as FC histograms or as vertical bars with either individual values (in K), or with mean ± SEM. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001) and correspond to two-tailed non-parametric Mann–Whitney test. The data represent a minimum of three independent experiments. Source data are available for this figure: SourceData FS1.$

The platelet purity, gating strategy, and human NSCLC platelet assays. (A) Representative contour or pseudocolor plots for gating strategy (left) and platelet purity (CD41⁺) are shown for FC assays performed on washed platelets. (B) The FC histogram overlay showing washed platelets stained with anti–TLT-1 (solid blue) or its isotype control (unfilled blue) and the unstained control sample in solid gray. (C) The quantitation (mean ± SEM) of TLT-1–relative mRNA normalized to β-tubulin in individual samples. Statistical significance tested by Mann–Whitney test. (D) Quantitation of total TLT-1 platelet expression by Western blot. Statistical significance tested by Mann–Whitney test. (E) The FC histograms overlay for positive control for PAC1 marker where washed healthy platelets were stained for PAC1 after activation with ADP (green) or without any external stimulation (blue). The signal from unstained platelets is shown in gray. The events (30,000) were acquired at low flow rate and data analyzed by FlowJo. (F and G) Representative FC pseudocolor plots of washed platelets from NSCLC patients and controls show the NAO-stained platelets where NAO-ve population represents ex vivo apoptotic platelets with their quantitation (G; n = 15 for control and n = 24 for NSCLC). The assay measures the loss of mitochondrial integrity by measuring NAO fluorescence loss from platelets. Statistical significance tested by Mann–Whitney test. (H and I) Representative Western blot–based analysis (H) of total platelet expression of activated Caspase 3, Bax, and Bcl2 proteins in NSCLC and control and their quantitation (I) normalized to loading control. The band densities were measured by ImageJ software and normalized to β-tubulin (loading control). (J) Quantitation of PMV expressing TLT-1 as measured by FC. Statistical significance tested by Mann–Whitney test. (K) ELISA-based comparison of quantitation of TLT-1 in isolated microvesicle fraction versus soluble fraction representing microvesicle-depleted plasma. Each bar represents one patient or control sample as indicated. The data in this figure are presented as FC histograms or as vertical bars with either individual values (in K), or with mean ± SEM. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001) and correspond to two-tailed non-parametric Mann–Whitney test. The data represent a minimum of three independent experiments. Source data are available for this figure: SourceData FS1.

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