Fig 1: PSPC1 expression and its cleavage in patients with OSA. (a) The mean fluorescence intensity (MFI) of the monocyte PSPC1 intracellular expression (as determined by flow cytometry) in HV (n = 8) and the patients with severe OSA (n = 15) are shown. The PSPC1 distribution (left panel) and PSPC1 histogram (right panel) are shown; (b) The monocytes PSPC1 mRNA expression (estimated by qPCR) in the monocytes from HV (n = 23) and the patients with severe OSA (n = 50) are shown; (c) The PSPC1 plasma protein was quantified using ELISA (n = 18 HV and n = 45 patients with severe OSA). The intergroup differences in PSPC1 expression were assessed using an unpaired t-test with Welch’s test correction. Error bars: Standard error of the mean (SEM). * p < 0.05, ** p < 0.01 when compared with HV; (d) Correlation between PSPC1 and TGFß mRNA expression in the monocytes from the patients with severe OSA (n = 45) (left panel) and the correlation between PSPC1 and TGFß protein concentration in the plasma of the patients with severe OSA (n = 40) (right panel). The patients were randomly selected. Spearman correlation coefficients (r) and p-values are shown; (e) Correlation between MMP2 and PSPC1 mRNA expression in the monocytes from the patients with severe OSA (n = 45) (left panel) and the correlation between MMP2 and PSPC1 protein concentration in the plasma of the patients with severe OSA (n = 40) (right panel). Pearson’s correlation coefficients (r) and p-values are shown; (f) MMP2 mRNA expression analysis by qPCR in the monocytes from HV (n = 18) and the patients with severe OSA (n = 45) (left panel). The MMP2 protein was quantified using ELISA (n = 20 HV, and n = 40 patients with OSA) (right panel). The groups were compared using an unpaired t-test with Welch’s test correction. Error bars: SEM. * p < 0.05, **** p < 0.0001 as compared with HV; (g) The PSPC1 expression by flow cytometry analysis is shown. Monocytes from HV (n = 3) were cultured under normoxia or IH for 24 h and then treated with MMP2 for 3 h (left and middle panels). Supernatants were collected to evaluate PSPC1 protein concentrations using ELISA (right panel). Mean values are shown. Spearman coefficients (r) and p-values (p) were calculated for the left panel N (r = 0.3143, p = 0.5639) and IH (r = -0.02, p = >0.99) and right panel N (r = -0.8286, p = 0.0583) and IH (r = -1.00, p = 0.0028); (h) Western blot analysis of enzymatic digestion of PSPC1 with or without MMP2 (molar ratio of 1:10) is shown (left panel). The expected size of 59 kDa (uncleaved) and an additional band of approximately 43 kDa (cleaved) were observed. Three different experiments were performed with similar findings (right panel). The comparison was performed by unpaired t-test; ** p < 0.01.
Fig 2: Hypoxemia collaborates with PSPC1 to modulate TGFß expression. The melanoma cell line (C-8161) was cultured under N and IH conditions with rPSPC1 protein (2.5 ng/mL) with or without a-PSPC1 (1 ug/mL) for 16 h. The melanoma cells were then harvested to analyze TGFß mRNA expression by qPCR. The groups were compared using two-way ANOVA. Error bars: SEM. * p < 0.01, ** p < 0.001 are shown.
Fig 3: Effect of PSPC1 on EMT-TFs expression in a melanoma cell line. The melanoma cell line (C-8161) was cultured under N and IH conditions with rPSPC1 protein (2.5 ng/mL) with or without a-PSPC1 (1 ug/mL) for 16 h. The melanoma cells were then harvested to analyze TWIST (a), SLUG (b), and SNAIL (c) mRNA expression by qPCR. The groups were compared using a two-way ANOVA. Error bars: SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The melanoma cell line (C-8161) was cultured under IH and N conditions with plasma from randomly selected patients with severe OSA (10% concentration) for 16 h. The melanoma cells were then harvested to analyze TWIST, SLUG, and SNAIL mRNA expression by qPCR analysis. The correlation between PSPC1 protein concentrations from OSA plasma supplemented to the cell culture with mRNA expression of EMT-TFs in N (d) and IH (e) conditions are shown. The correlation between TGFß protein concentrations from OSA plasma supplemented to the cell culture and mRNA expression of EMT-TFs in N (f) and IH (g) conditions are shown. Pearson’s correlation coefficients (r) and p-values are shown.
Fig 4: HIF1a is associated with PSPC1. (a) Correlation between PSPC1 and HIF1a mRNA expression in monocytes in patients with OSA (n = 40). Pearson correlation coefficients (r) and p-values are shown; (b) Monocytes from HV (n = 7) were treated with 30 µM of PX478 and/or exposed to IH for 16 h. Paired control samples were incubated under normoxia conditions and without PX478. The distribution estimation of MFI of the PSPC1 intracellular expression (as determined by flow cytometry, left panel) and the PSPC1 histogram (right panel) are shown; (c) PSPC1 mRNA expression estimated by qPCR in the monocytes from HV (n = 7) treated with a specific HIF1a inhibitor (30 µM of PX478) or not and exposed to IH or normoxia conditions for 16 h (left panel), monocytes from HV (n = 3) treated with or without siHIF1a and exposed to IH or normoxia conditions for 16 h (middle panel), monocytes from HV (n = 3) treated or not with DMOG for 2 h (right panel); (d) Correlation between MMP2 and HIF1a mRNA expression in the monocytes from the patients with severe OSA (n = 40). Spearman’s correlation coefficients (r) and p-values are shown; (e) MMP2 mRNA expression estimated by qPCR in the monocytes from HV (n = 7) treated with a specific HIF1a inhibitor (30 µM of PX478) or not and exposed to IH or normoxia conditions for 16 h (left panel), monocytes from HV (n = 3) treated with or without siHIF1a and exposed to IH or normoxia conditions for 16 h (middle panel), monocytes from HV (n = 3) treated or not with DMOG for 2 h (right panel). The groups were compared with a Two-way ANOVA or paired t-test. Error bars: SEM. * p < 0.05; ** p < 0.01, *** p < 0.001, compared with the untreated cells. Spearman’s correlation coefficients (r) and p-values are shown.
Fig 5: PSPC1 expression in patients with OSA increases TGFß expression effect on EMT-TFs. Intermittent hypoxia increases the activation of HIF1a, which might bind with the promoter of genes such as PSPC1, MMP2, and TGFß, leading to high levels of these proteins in severe OSA monocytes. The MMP2 plays a role in PSPC1 cleavage, increasing plasma PSPC1 levels. The combination of high plasma levels of TGFß and PSPC1 increases the EMT-TF effect through TWIST and SLUG and CSC effect by SOX2 in melanoma cells.
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