Journal: Cell Death & Disease

Article Title: Notch-Hes-1 axis controls TLR7-mediated autophagic death of macrophage via induction of P62 in mice with lupus

doi: 10.1038/cddis.2016.244

Figure Lengend Snippet: P62/SQSTM1 is requisite for TLR7-induced autophagic death of macrophages. ( a ) RAW 264.7 cells were unstimulated or stimulated with R848 (0.1 μ g/ml) for 6 h. Real-time PCR was carried out to examine mRNA levels of the five autophagic adaptors in RAW 264.7 cells. ( b ) P62 expression was examined by real-time PCR in RAW 264.7 cells after R848 (0.1 μ g/ml) stimulation. ( c ) Western analysis of P62 expression in RAW 264.7 cells treated with R848 (0.1 μ g/ml) for indicated time. ( d , f ) RAW 264.7 cells were transfected with si-control or si-P62 for 24 h and then stimulated with R848 for 24 h. si-P62#1 and si-P62#2 represent two independent siRNAs targeting P62. ( d ) Protein level of LC3II was detected by western analysis. ( f ) The percentage of total dead RAW 264.7 cells was detected by flow cytometry. Total dead RAW 264.7 cells included Annexin V+ PI+, Annexin V+PI−and Annexin V– PI+ cells. Representative FACS dot plots were shown in the left. ( e ) Degree of knockdown of P62 in samples used in ( d ). ( a , b , f ) The data are shown as means±S.E.M.s and represent three independent experiments. * P <0.05, ** P <0.01, determined by t- tests

Article Snippet: The small-interfering RNA used for silencing mouse P62 and Hes-1 were obtained from Ribobio (Guangzhou, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Control, Flow Cytometry, Knockdown

Journal: Cell Death & Disease

Article Title: Notch-Hes-1 axis controls TLR7-mediated autophagic death of macrophage via induction of P62 in mice with lupus

doi: 10.1038/cddis.2016.244

Figure Lengend Snippet: Hes-1 regulates TLR7-induced autophagic death of macrophages via P62. ( a ) RAW 264.7 cells were pre-treated with DAPT (10 μ M) or DMSO (0.02%) for 2 h and then stimulated by R848 (0.1 μ g/ml) for 24 h. Protein level of LC3II was assessed by western blot. ( b ) Degree of knockdown of Hes-1 in samples used in ( c ). ( c , f ) RAW 264.7 cells were transfected with si-control or si-Hes-1 for 24 h and then stimulated with R848 for 24 h. si-Hes-1#1 and si-Hes-1#2 represent two independent siRNAs targeting Hes-1. ( c ) Protein levels of Hes-1, P62 and LC3II were detected by western blot and normalized to GAPDH. ( f ) The percentage of total dead RAW 264.7 cells was detected by flow cytometry. Total dead RAW 264.7 cells included Annexin V+ PI+, Annexin V+PI− and Annexin V− PI+ cells. Representative FACS dot plots were shown (left). ( d ) PEX-2 plasmid transfected or PEX-2-Hes-1 plasmid transfected RAW 264.7 cells were stimulated with R848 (0.1 μ g/ml) for 24 h. Protein expression levels of Hes-1, P62 and LC3II were detected by western blot and normalized to GAPDH. ( e ) mRNA level of P62 in RAW 264.7 cells transfected with PEX-2-Hes-1 plasmid or control vector for indicated time. ( e , f ) The data are shown as means±S.E.M.s and represent three independent experiments. * P <0.05, ** P <0.01, determined by t- tests

Article Snippet: The small-interfering RNA used for silencing mouse P62 and Hes-1 were obtained from Ribobio (Guangzhou, China).

Techniques: Western Blot, Knockdown, Transfection, Control, Flow Cytometry, Plasmid Preparation, Expressing

Journal: Cell Death & Disease

Article Title: Notch-Hes-1 axis controls TLR7-mediated autophagic death of macrophage via induction of P62 in mice with lupus

doi: 10.1038/cddis.2016.244

Figure Lengend Snippet: Autophagic death of macrophage is re-verified in TLR7 agonist-induced lupus mouse model. TLR7 agonist-induced lupus mouse model was established by treating wild-type C57BL/6 mice with imiquimod for 10 weeks. ( a ) The percentage of F4/80+ macrophages among total splenic cells in IMQ-treated mice ( n =8) and control mice ( n =9) was determined by flow cytometry analysis. ( b ) Expression levels of macrophage activation markers (CD80, CD86 and MHCII) in splenic macrophages from IMQ-treated mice ( n =8) and control mice ( n =9). ( c ) The proportion of F4/80+ Annexin V+ macrophages among total splenic cells in IMQ-treated mice ( n =8) and control mice ( n =9). ( d , g , i , j ) Flow cytometry analysis of the levels of LC3IIB ( d ), P62 ( g ), Notch1 ( i ) and Hes-1 ( j ) in splenic macrophages from IMQ-treated mice ( n =8) and control mice ( n =9). Representative histograms showed the MFI of detected proteins were all on the right of stastical charts. ( e , h ) BMDM cells were generated from the bone marrow of IMQ-treated mice ( n =5) and control mice ( n =5). The protein levels of LC3II ( e ) and P62 ( h ) were assessed by western blot and normalized to GAPDH. ( f ) The correlation analysis between percentage of F4/80+ Annexin V+ splenic macrophages and MFI of LC3II in splenic macrophages from IMQ-treated mice. And the correlation results were determined by linear regression analysis. ( a , c , d , g , i , j ) The data are shown as means±S.E.M.s and represent two independent experiments. ** P <0.01, *** P <0.001, determined by t -tests

Article Snippet: The small-interfering RNA used for silencing mouse P62 and Hes-1 were obtained from Ribobio (Guangzhou, China).

Techniques: Control, Flow Cytometry, Expressing, Activation Assay, Generated, Western Blot

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Study flow diagram. UVM/UM, uveal melanoma; TCGA, The Cancer Genome Atlas; TME, tumor microenvironment; LASSO, least absolute shrinkage and selection operator; PRRX1, paired related homoeobox 1; ROC, receiver operating characteristic; WB, Western blot; siRNA, small interfering RNA; GSEA, Gene Set Enrichment Analysis; GEO, Gene Expression Omnibus; TMB, tumor mutation burden.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Selection, Western Blot, Small Interfering RNA, Gene Expression, Mutagenesis

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: The overall prognostic performance of PRRX1 expression in two UM cohorts. Kaplan–Meier analyses of (A) OS in TCGA cohort and (B) MFS in the GEO cohort, the numbers shown below the survival curves are the number of patients at risk at the specified year. Distribution of PRRX1 expression levels in the (C) TCGA and (D) GEO cohorts, the dotted line represented the median PRRX1 expression and divided the patients into low-expression (green dots) and high-expression groups (red dots). Percentages of (E) deceased patients (red dots) in the TCGA cohort and (F) patients with metastasis (red dots) in the GEO cohort as a function of PRRX1 expression level.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Prognostic predictive ability of PRRX1 expression. (A) Univariate and multivariate Cox analyses of PRRX1 expression and seven other clinicopathological parameters in the TCGA cohort. (B) Time-dependent ROC analysis for prediction of OS in the TCGA cohort. (C) ROC analyses of PRRX1 expression and other seven clinicopathological parameters for predicting OS in the TCGA cohort. (D) Univariate and multivariate Cox analyses of PRRX1 expression and six other clinicopathological parameters in the GEO cohort. (E) Time-dependent ROC analysis for prediction of MFS in the GEO cohort. (F) ROC analyses of PRRX1 expression and other six clinicopathological parameters for predicting MFS in the GEO cohort. Corresponding AUC of each curve is displayed in the lower right corner of each figure, the higher the AUC value, the better the predictive power of the parameter.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: (A) Association between PRRX1 expression level and OS in patients with 33 types of cancer from the TCGA database, red dots represent the possible risk factors while blue represents potential protective factors, abbreviations for all cancer types are in Table S4 . (B) Correlations between PRRX1 expression and chromosome 3/8q copy number, the density curve on the x and y-axes represents the distribution trend of themselves. The blue line indicates the best fitted linear models and the shaded area indicates the 95% confidence interval. (C) GSEA plot of the top 20 ranked Hallmark sets of genes associated with high PRRX1 expression, as determined by comparing high and low PRRX1 expression groups (FDR < 0.25, p < 0.05).

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Analysis of immunologic characteristics associated with PRRX1 expression levels. (A) Comparisons of the levels of expression of immune checkpoint genes in the high and low PRRX1 groups (** p < 0.01; *** p < 0.001). (B) Correlation between PRRX1 expression level and TMB. (C) Correlation between PRRX1 expression and tumor immune/stromal cell populations in the UM microenvironment: T cells, CD8 T cells, cytotoxic lymphocytes, B lineage cells, NK cells, cells of the monocytic lineage, myeloid dendritic cells, neutrophils, endothelial cells, and fibroblasts, the x-axis represents the expression levels of PRRX1 , and the y axis is the abundance of TME cells. The density curve on the x and y-axes represents the distribution trend of themselves. The blue line indicates the best fitted linear models and the shaded area indicates the 95% confidence interval.

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PRRX1 Is a Novel Prognostic Biomarker and Facilitates Tumor Progression Through Epithelial–Mesenchymal Transition in Uveal Melanoma

doi: 10.3389/fimmu.2022.754645

Figure Lengend Snippet: Knockdown of PRRX1 in UM cell suppresses EMT, migration and invasion in vitro . (A) GSEA results revealed that PRRX1 expression significantly positively correlated with the EMT signatures (NES = 1.6, p < 0.001, FDR = 0.002). (B) WB showed PRRX1 protein level with three different PRRX1 –siRNA treatment, relative expression levels of proteins were normalized based on the β-actin levels. (C) The influence of PRRX1 knockdown on the expression level of EMT-related factors (Snail, N-cadherin and E-cadherin) were analyzed using WB, relative expression levels of proteins were normalized based on the β-actin levels (left: representative images of three independent experiments; right: quantitative analyses, n = 3, * p < 0.05; ** p < 0.01; *** p < 0.001, paired Student’s t-test). (D) Wound-healing assay was used to detect cell migration ability in si-NC and si- PRRX1 UM cells (left: representative images of three independent experiments; right: quantitative analyses, n = 3, * p < 0.05, paired Student’s t-test). (E) Transwell assays showed the effect of PRRX1 knockdown on UM cell invasion (left: representative images of three independent experiments; right: quantitative analyses, n = 3, *** p < 0.001, unpaired Student’s t-test).

Article Snippet: PRRX1 small interfering (si) RNA (si- PRRX1 ) and negative control (si-NC) oligonucleotide were purchased from RiboBio (Ribobio, Guangzhou, China) with the sequences GGAATAGGACAACCTTCAA (Si1), ACACTATCCTGATGCTTTT (Si2), and GTTCCGCAGGAATGAGAGA (Si3).

Techniques: Knockdown, Migration, In Vitro, Expressing, Wound Healing Assay