Journal: Oncology reports

Article Title: Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma.

doi: 10.3892/or.2017.5785

Figure Lengend Snippet: Figure 1. The expression of PRRX1 and p53 in HCC tissues and cell lines. (A) Representative immunohistochemistry images of human HCC tissues for detec- tion of PRRX1 and p53 (magnification, x100). (B) Protein expression of PRRX1 and p53 in HCC tissues and adjacent liver tissues were detected by western blotting. GAPDH was used as a control. (C) Protein expression of PRRX1 and p53 in HCC cell lines and normal liver cells. GAPDH was used as a control.

Article Snippet: Slides were incubated in monoclonal antibodies against goat polyclonal anti-PRRX1 (NBP1-06067, 1:50 dilutions; Novus Biologicals LLC, Littleton, CO, uSA), rabbit monoclonal anti-p53 (ab179477, 1:100 dilutions; Abcam, Cambridge, uK) at 4 ̊C overnight, followed by incubation in the corresponding secondary antibodies at 37 ̊C for 30 min. Staining was performed with DAB and counterstaining with Mayer's hematoxylin.

Techniques: Expressing, Immunohistochemistry, Western Blot, Control

Journal: Oncology reports

Article Title: Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma.

doi: 10.3892/or.2017.5785

Figure Lengend Snippet: Figure 2. Decreased expression of PRRX1 promotes apoptosis resistance, migration and invasion of HCC cells by regulating p53 expression. (A) Expression of apoptosis-related protein in SMMC-7721 and HepG2 cultured with PRRX1-siRNA, detected by western blotting. GAPDH was used as a control. (B and C) Wound healing assays and transwell assays show increased migration and invasion ability of HCC cells by using PRRX1-siRNA. The numbers of invasive HCC cells were calculated of ten random microscopic fields. Data are shown as the mean ± SD of three independent experiments (*P<0.05).

Article Snippet: Slides were incubated in monoclonal antibodies against goat polyclonal anti-PRRX1 (NBP1-06067, 1:50 dilutions; Novus Biologicals LLC, Littleton, CO, uSA), rabbit monoclonal anti-p53 (ab179477, 1:100 dilutions; Abcam, Cambridge, uK) at 4 ̊C overnight, followed by incubation in the corresponding secondary antibodies at 37 ̊C for 30 min. Staining was performed with DAB and counterstaining with Mayer's hematoxylin.

Techniques: Expressing, Migration, Cell Culture, Western Blot, Control

Journal: Oncology reports

Article Title: Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma.

doi: 10.3892/or.2017.5785

Figure Lengend Snippet: Figure 4. Depletion of PRRX1 and p53 affects HCC cell apoptosis. Interfering with the expression of PRRX1 or p53 inhibits apoptosis of HCC cells. Both decreased expression of PRRX1 and p53 induced stronger resistance to apoptosis of HCC cells (*P<0.05, **P<0.01).

Article Snippet: Slides were incubated in monoclonal antibodies against goat polyclonal anti-PRRX1 (NBP1-06067, 1:50 dilutions; Novus Biologicals LLC, Littleton, CO, uSA), rabbit monoclonal anti-p53 (ab179477, 1:100 dilutions; Abcam, Cambridge, uK) at 4 ̊C overnight, followed by incubation in the corresponding secondary antibodies at 37 ̊C for 30 min. Staining was performed with DAB and counterstaining with Mayer's hematoxylin.

Techniques: Expressing

Journal: Oncology reports

Article Title: Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma.

doi: 10.3892/or.2017.5785

Figure Lengend Snippet: Figure 3. Depletion of PRRX1 and p53 enhances migration and invasion ability of HCC cells. Transwell assays show that downregulation of both PRRX1 and p53 present increased migration and invasion ability of HCC cells compared with PRRX1-siRNA or p53-siRNA alone.

Article Snippet: Slides were incubated in monoclonal antibodies against goat polyclonal anti-PRRX1 (NBP1-06067, 1:50 dilutions; Novus Biologicals LLC, Littleton, CO, uSA), rabbit monoclonal anti-p53 (ab179477, 1:100 dilutions; Abcam, Cambridge, uK) at 4 ̊C overnight, followed by incubation in the corresponding secondary antibodies at 37 ̊C for 30 min. Staining was performed with DAB and counterstaining with Mayer's hematoxylin.

Techniques: Migration

Journal: Oncology reports

Article Title: Downregulation of PRRX1 via the p53-dependent signaling pathway predicts poor prognosis in hepatocellular carcinoma.

doi: 10.3892/or.2017.5785

Figure Lengend Snippet: Figure 5. Decreased expression of PRRX1 and p53 is associated with poor prognosis in HCC patients. Overall survival rate and disease-free survival rate in 116 patients who underwent resection for primary HCC based on PRRX1 and p53 expression. Both downregulated expression of PRRX1 and p53 are associated with a poorer overall and disease-free survival rate (P<0.001).

Article Snippet: Slides were incubated in monoclonal antibodies against goat polyclonal anti-PRRX1 (NBP1-06067, 1:50 dilutions; Novus Biologicals LLC, Littleton, CO, uSA), rabbit monoclonal anti-p53 (ab179477, 1:100 dilutions; Abcam, Cambridge, uK) at 4 ̊C overnight, followed by incubation in the corresponding secondary antibodies at 37 ̊C for 30 min. Staining was performed with DAB and counterstaining with Mayer's hematoxylin.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: NLRP3 Regulates Mandibular Healing through Interaction with UCHL5 in MSCs

doi: 10.7150/ijbs.78174

Figure Lengend Snippet: NLRP3 inflammasome activation in MSC-mediated mandibular healing. Two-month-old WT or Prx1-Cre/ROSA nTnG mice were used, as specified in the figure legends. Mice received mandibular osteotomy surgery and were sacrificed at various time points during healing, N=4. (A) Representative images of ALP-stained paraffin sections show mandible defects of WT mice. ALP-positive area relative to tissue area (%) was determined. (B) Representative images of TRAP-stained paraffin sections show defect areas of WT mice. The surface of osteoclasts relative to the bone surface (%) was determined. (C) Representative images of unstained frozen sections under fluorescence microscopy and H&E-stained adjacent sections of mandible defects at POD 14 of Prx1-Cre/ROSA nTnG mice. Images show Prx1-Cre + cells (GFP, green) and Cre - cells (tdTomato, red) in defect areas. Prx1-Cre + or Cre - cell number relative to all cell number (%) was assessed. (D) Representative IHC images for NLRP3 in defect areas at various time points. The percentage of NLRP3-positive area was calculated. (E) Paraffin sections of WT mice at POD 7 were subjected to double immunofluorescence staining with anti-Prx1 and anti-NLRP3. Representative images of defect areas are shown. The proportion of NLPR3 + Prx1 + or NLPR3 + Prx1 - area to NLRP3 + area (%) were determined. (F-H) M-MSCs from WT mice were cultured and stimulated with or without 10 μg/ml LPS ± 5 mM ATP during culture, as indicated in the figures. (F) Western blot of cell lysates and supernatants from M-MSCs cultures. (G) The relative gene expression levels of Col1α and OCN were determined by qPCR. (H) M-MSCs were cultured in osteoblast-inducing medium for 7 days. Culture plates were stained for ALP, and ALP + areas were measured. Scale bars, 200 μm (A, B and D) and 100 μm (C and E). All error bars represent SEM. Two-tailed unpaired Student's t-test was performed. * P < 0.05 vs. POD 0 or in the indicated groups.

Article Snippet: The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H 2 O 2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C.

Techniques: Activation Assay, Staining, Fluorescence, Microscopy, Double Immunofluorescence Staining, Cell Culture, Western Blot, Expressing, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: NLRP3 Regulates Mandibular Healing through Interaction with UCHL5 in MSCs

doi: 10.7150/ijbs.78174

Figure Lengend Snippet: NLRP3 deficiency promotes osteoblast differentiation and mandibular healing. Two-month-old WT, NLRP3 KO , or Prx1-Cre/ROSA nTnG mice were used. (A) Schematic of experimental design. (B-D) Frozen sections of defect areas from Prx1-cre/Rosa nTnG mice received locol injection of MCC950 or PBS were used, N=4. (B) Representative images of unstained frozen sections under fluorescence microscopy and H&E-stained adjacent sections of mandible defects at POD 14 of Prx1-Cre/ROSA nTnG mice. Prx1-Cre + area (%) was assessed. The number of Prx1-Cre + cells along the bone surface relative to the number of total Prx1-Cre + cells (%) was determined. (C) Representative images of ALP-stained frozen sections show mandible defects. ALP-positive area relative to tissue area (%) was determined. (D) Representative reconstructed sections of mandibles (upper panels) and defects (lower panels) and morphometric data of bone volume (mm 3 ) relative to tissue volume (%). (E) Primary cultures of mandibular bone marrow cells from WT and NLRP3 KO mice were stained with methylene blue to show total CFU-F colonies, stained cytochemically for ALP to detect CFU-ALP + colonies, or stained with Alizarin Red to show mineralized nodule formation. The number of CFU-F colonies, CFU-ALP + colonies and nodules per well (#/well) were counted. (F-I) Two-month-old WT and NLRP3 KO mice received mandibular osteotomy surgery and were sacrificed at various time points during healing, N=6. (F) Representative reconstructed sections of mandibles (upper panels) and defects (lower panels). (G) Morphometric data of bone volume (mm 3 ) relative to tissue volume (%). (H) Representative images of ALP-stained paraffin sections show mandible defects. (I) ALP-positive area relative to tissue area (%) was determined. Scale bars, 200 μm. All error bars represent SEM. Two-tailed unpaired Student's t-test was performed. * P < 0.05 in the indicated groups.

Article Snippet: The deparaffinized sections were subjected to heat mediated antigen retrieval, and blocked in H 2 O 2 for 30 minutes, followed by PBS with 5% BSA and 0.2% Triton X-100 at room temperature for 30 minutes, and then stained overnight with primary antibody against NLRP3 (Abcam, Cat#ab214185), Prx1 (Origene, Cat#TA803116), USP1 (CST, Cat#D37B4) or UCHL5 (Abcam, Cat#ab133508) at 4 °C.

Techniques: Injection, Fluorescence, Microscopy, Staining, Two Tailed Test

Journal: iScience

Article Title: In vitro and in vivo models define a molecular signature reference for human embryonic notochordal cells

doi: 10.1016/j.isci.2024.109018

Figure Lengend Snippet: Transcriptomic analyses at single-cell resolution of hiPSC differentiation into notochordal-like cells (A) Unsupervised clustering of iPS-NLC single-cell dataset revealing 9 lineage-associated clusters. Clusters of primitive-streak (PS), neuromesodermal progenitors (NMP), lateral/paraxial mesoderm progenitors (L/PXM Prog.), axial progenitors (Axial Prog.), cardiac mesoderm (Cardiac Mes.), notochord, endoderm, neuroectoderm (Neuroect.) are projected on UMAP embedding. Values indicate the number of cells per cluster. (B) Distribution of cells co-expressing TBXT, SHH, NOG, CHRD, SOX9, FOXA2 notochord-related markers on UMAP embedding. Expression scores, displayed from lowest (gray) to highest (red), were calculated using the AddModuleScore function. (C) Distribution of day 7 differentiated cells related to their treatment on UMAP embedding. Treatments day 7 not transfected (d7 NT), day 7 NOTO transfected (d7 NOTO) and day 7 NOTO treated with SB (d7 NOTO SB) are plotted with a distinct color code for iPS-NLC dataset. Note that although the two clusters of primitive-streak have similar transcriptomic profile they are distinct in their composition “NT”/“NOTO”. (D) Cell type differentiation profile related to treatment for two representative hiPSC lines. Normalized values using total number of cells analyzed per treatment in iPS-NLC dataset are shown in %, for day 7 not transfected (d7 NT), day 7 NOTO transfected (d7 NOTO) and day 7 NOTO treated with SB (d7 NOTO SB), for hiPSC a and hiPSC b . (E) Inhibition of Tgf-β signaling pathway significantly enhances NLC markers expression and reduces off-target differentiation at day 7. Relative expression of notochord (endogenous mRNA for NOTO and TBXT ), cardiac ( HAND1, PRRX1 ) and endoderm ( GATA4, SOX17 ) markers by RT-qPCR. Mean expression levels are presented as fold changes relative to unstimulated hiPSC and standard error is indicated, for n = 14 biological replicates using hiPSC a,b,c,d,e . Dotted line represents the unstimulated hiPSC expression level. Statistical significance was measured by a student test for each gene marker. ∗∗p < 0.001, ∗∗∗p < 0.0001. (F) Dynamic velocities projected into UMAP-embedding revealing differentiation trajectories. The spliced and unspliced mRNA dynamics were inferred using scVelo, projected into low dimension space with veloAE and visualized here by arrows. Cells are colored based on cluster assignation. (G) Identification of major WGCNA modules of co-expressed genes distinguishing cell clusters. Each row represents a WGCNA module, and each column comprises all cells from the annotated clusters. The height of rows indicates the number of genes in the module and the expression level is shown colored from lowest (blue) to highest (red). Three selected lineage-associated genes co-expressed within modules are indicated ordered by intra-connectivity. (H) Violin plots of selected genes differentially expressed in NO cluster and their corresponding tissue specificities in various species described in the literature. Genes are ordered by Log2FC. Star indicates their belonging to the NO specific WGCNA module. Table on the left recapitulates genes links to lineage, developmental or post-natal time frame and species information curated from the literature. mouse (M), human (H), bovine (B), dog (D), xenopus (X), chick (C), zebrafish (Z). Abbreviations Fig2: AXP: axial progenitors; CM: cardiac mesoderm; END: endoderm; L/PXP: lateral/paraxial mesoderm progenitors; MEPC: mesendoderm progenitors; NE: neuroectoderm; NLC: notochordal-like cells; NMP: neuromesodermal progenitors; NO: notochord; PS: primitive-streak.

Article Snippet: TaqMan probe PRRX1 , Life Technologies , Hs00246567_m1.

Techniques: Expressing, Transfection, Inhibition, Quantitative RT-PCR, Marker

Journal: iScience

Article Title: In vitro and in vivo models define a molecular signature reference for human embryonic notochordal cells

doi: 10.1016/j.isci.2024.109018

Figure Lengend Snippet:

Article Snippet: TaqMan probe PRRX1 , Life Technologies , Hs00246567_m1.

Techniques: Recombinant, Membrane, Transfection, Passaging, Modification, Knock-Out, Sample Prep, SYBR Green Assay, Software