Figure 2.
RIPK3 inhibition reduces TNF levels in MF supernatant and increases the viability of MF PBMCs. (A) Cytokine levels of IL-8 were determined by CBA in the PB serum of 3 healthy control subjects and 1 patient with MF. Data are shown as dot plots reporting mean and standard deviation. P values from Mann-Whitney test as indicated in the figure. (B) Cytokine levels of VEGF were determined by CBA in the PB serum of 3 healthy control subjects and 20 patients with MF. Data are shown as dot plots reporting mean and standard deviation. P values from Mann-Whitney test as indicated in the figure. (C) RIPK3 protein levels were quantified in PBMCs from 14 patients with MF as described in Figure 1A and correlated with corresponding b-FGF levels (pg/mL) analyzed by CBA. The strength of the association was calculated by Pearson correlation coefficient. The functional relationship was characterized by linear regression analysis. Pearson r = 0.5548, P = .0488, y = 7.301X -17.37. (D) Cytokine levels of TNF were determined by CBA in the supernatant of liquid culture of PBMCs from 6 patients with MF, and 4 healthy control subjects after treatment with the RIPK3 inhibitor GSK’843 (1 μM) and vehicle control DMSO (1:1000) for 72 hours. Data are shown as dot plots reporting mean and standard deviation. Student t test as shown in the figure. (E) Cell viability of PBMCs from 3 healthy control subjects and 4 patients with MF was analyzed by flow cytometry after staining with 7AAD and Annexin V. Shown is the ratio between the percentage of viable PBMCs after treatment with soluble control (DMSO 1:1000), and the percentage of viable PBMCs after treatment with GSK’843 (1 μM) for 72 hours. Shown are the mean and error bars denoting standard deviation. For comparison between entities, one-way ANOVA was performed (P = .0037) with a post-hoc Tukey test as shown in the figure. For comparison between treatment (GSK 1 μM) and soluble control (DMSO 1:1000), Student t test was used as shown in the figure. (F) BMMCs were treated for 72 hours with GSK’843 (1 μM) and vehicle control (DMSO 1:1000), before being plated in cytokine-enriched methylcellulose. The number of colonies was counted after 14 days. Experiments were performed in duplicates. Shown is the number of colonies from 3 patients with pre-MF (including pre-MF and PMF) and 4 healthy control subjects. The colony subtypes are indicated in the legend. Two-way ANOVA was performed. All panels: level of significance: .05; ∗P < .05, ∗∗P < .005. ANOVA, analysis of variance; BMMCs, BM mononuclear cells; CBA, cytometric bead assay; IL-8, interleukin-8; PMF, primary myelofibrosis; VEGF, vascular endothelial growth factor.

RIPK3 inhibition reduces TNF levels in MF supernatant and increases the viability of MF PBMCs. (A) Cytokine levels of IL-8 were determined by CBA in the PB serum of 3 healthy control subjects and 1 patient with MF. Data are shown as dot plots reporting mean and standard deviation. P values from Mann-Whitney test as indicated in the figure. (B) Cytokine levels of VEGF were determined by CBA in the PB serum of 3 healthy control subjects and 20 patients with MF. Data are shown as dot plots reporting mean and standard deviation. P values from Mann-Whitney test as indicated in the figure. (C) RIPK3 protein levels were quantified in PBMCs from 14 patients with MF as described in Figure 1A and correlated with corresponding b-FGF levels (pg/mL) analyzed by CBA. The strength of the association was calculated by Pearson correlation coefficient. The functional relationship was characterized by linear regression analysis. Pearson r = 0.5548, P = .0488, y = 7.301X -17.37. (D) Cytokine levels of TNF were determined by CBA in the supernatant of liquid culture of PBMCs from 6 patients with MF, and 4 healthy control subjects after treatment with the RIPK3 inhibitor GSK’843 (1 μM) and vehicle control DMSO (1:1000) for 72 hours. Data are shown as dot plots reporting mean and standard deviation. Student t test as shown in the figure. (E) Cell viability of PBMCs from 3 healthy control subjects and 4 patients with MF was analyzed by flow cytometry after staining with 7AAD and Annexin V. Shown is the ratio between the percentage of viable PBMCs after treatment with soluble control (DMSO 1:1000), and the percentage of viable PBMCs after treatment with GSK’843 (1 μM) for 72 hours. Shown are the mean and error bars denoting standard deviation. For comparison between entities, one-way ANOVA was performed (P = .0037) with a post-hoc Tukey test as shown in the figure. For comparison between treatment (GSK 1 μM) and soluble control (DMSO 1:1000), Student t test was used as shown in the figure. (F) BMMCs were treated for 72 hours with GSK’843 (1 μM) and vehicle control (DMSO 1:1000), before being plated in cytokine-enriched methylcellulose. The number of colonies was counted after 14 days. Experiments were performed in duplicates. Shown is the number of colonies from 3 patients with pre-MF (including pre-MF and PMF) and 4 healthy control subjects. The colony subtypes are indicated in the legend. Two-way ANOVA was performed. All panels: level of significance: .05; ∗P < .05, ∗∗P < .005. ANOVA, analysis of variance; BMMCs, BM mononuclear cells; CBA, cytometric bead assay; IL-8, interleukin-8; PMF, primary myelofibrosis; VEGF, vascular endothelial growth factor.

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