Journal: Cancers

Article Title: Intermittent Hypoxia Mediates Paraspeckle Protein-1 Upregulation in Sleep Apnea.

doi: 10.3390/cancers13153888

Figure Lengend Snippet: Figure 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 TGFβ mRNA expression in the monocytes from the patients with severe OSA (n = 45) (left panel) and the correlation between PSPC1 and TGFβ 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.

Article Snippet: Recombinant human MMP2 (R&D Systems) and PSPC1 were dissolved in 20-mM Tris buffer containing 2-mM CaCl2 and 1-mM MgCl2 (pH 7.3), at a concentration of 10 μg/100 L and 100 μg/100 μL, respectively.

Techniques: Expressing, Cytometry, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Protein Concentration, Cell Culture, Western Blot, Comparison

Journal: Cancers

Article Title: Intermittent Hypoxia Mediates Paraspeckle Protein-1 Upregulation in Sleep Apnea.

doi: 10.3390/cancers13153888

Figure Lengend Snippet: Figure 2. HIF1α is associated with PSPC1. (a) Correlation between PSPC1 and HIF1α 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 HIF1α 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 siHIF1α 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 HIF1α 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 HIF1α 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 siHIF1α 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.

Article Snippet: Recombinant human MMP2 (R&D Systems) and PSPC1 were dissolved in 20-mM Tris buffer containing 2-mM CaCl2 and 1-mM MgCl2 (pH 7.3), at a concentration of 10 μg/100 L and 100 μg/100 μL, respectively.

Techniques: Expressing, Control, Incubation, Cytometry

Journal: Cancers

Article Title: Intermittent Hypoxia Mediates Paraspeckle Protein-1 Upregulation in Sleep Apnea.

doi: 10.3390/cancers13153888

Figure Lengend Snippet: Figure 6. PSPC1 expression in patients with OSA increases TGFβ expression effect on EMT-TFs. Intermittent hypoxia increases the activation of HIF1α, which might bind with the promoter of genes such as PSPC1, MMP2, and TGFβ, 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 TGFβ and PSPC1 increases the EMT-TF effect through TWIST and SLUG and CSC effect by SOX2 in melanoma cells.

Article Snippet: Recombinant human MMP2 (R&D Systems) and PSPC1 were dissolved in 20-mM Tris buffer containing 2-mM CaCl2 and 1-mM MgCl2 (pH 7.3), at a concentration of 10 μg/100 L and 100 μg/100 μL, respectively.

Techniques: Expressing, Activation Assay, Clinical Proteomics

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing