Journal: Journal of Translational Medicine

Article Title: Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

doi: 10.1186/s12967-023-04066-x

Figure Lengend Snippet: PRDM1 and PRDM2 expression in CD4 + T lymphocyte activation. Bar graphs represent data from qRT-PCR analysis of PRDM1 and PRDM2 main transcripts ( BLIMP1a, BLIMP1b, RIZ1, RIZ2 ) in CD4 + T cells stimulated with anti-CD3/CD28 antibodies. PRDM1 and PRDM2 expression levels in CD4 + T cells were calculated using the ΔΔCt method with the indicated control gene. A schematic illustration of human PRDM1 and PRDM2 main products and used amplicons is also reported. To amplify PRDM1 two sets of primers, recognizing specific sequences of BLIMP1a and BLIMP1b transcripts, were used. PRDM2 gene expression was verified using two sets of primers recognizing sequences exclusive of RIZ1 or a common region to both RIZ1 and RIZ2 (and indicated as RIZex8 ). RIZ2 transcript was measured by subtraction as previously described . The ratio between the PR- and PR + transcripts for each gene was also calculated. The relative expression of activation marker genes CD40LG and IL2RA was also measured. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Western blot analysis was performed as described elsewhere [ ] with mouse monoclonal antibody FLAGTM clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Control, Gene Expression, Marker

Journal: Journal of Translational Medicine

Article Title: Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

doi: 10.1186/s12967-023-04066-x

Figure Lengend Snippet: Cytokine treatments regulate PRDM1 and PRDM2 gene expression levels in activated T cells. BLIMP1a, BLIMP1b, RIZ1, RIZex8 and RIZ2 gene expression analyses by qRT-PCR in activated T cells were compared to naïve T cells after treatment with IL2, IL4 and IFNA at 2 h and at 6 h. Three independent experiments in triplicates were performed and data expressed as mean ± SD. * P < 0.05 vs control cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Western blot analysis was performed as described elsewhere [ ] with mouse monoclonal antibody FLAGTM clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).

Techniques: Gene Expression, Quantitative RT-PCR, Control

Journal: Journal of Translational Medicine

Article Title: Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

doi: 10.1186/s12967-023-04066-x

Figure Lengend Snippet: Gene expression analysis of PRDM 1 and PRDM2 in Jurkat cells. On the left upper side, the basal levels of BLIMP1a, BLIMP1b, RIZ1, RIZex8 and RIZ2 in Jurkat cells are represented as fold on RPS18 control gene. The other bar graphs represent data from qRT-PCR analysis of the same transcripts in Jurkat cells following activation with PMA/Ion. Expression levels were calculated using the ΔΔCt method with the indicated control gene. Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Western blot analysis was performed as described elsewhere [ ] with mouse monoclonal antibody FLAGTM clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).

Techniques: Gene Expression, Control, Quantitative RT-PCR, Activation Assay, Expressing

Journal: Journal of Translational Medicine

Article Title: Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

doi: 10.1186/s12967-023-04066-x

Figure Lengend Snippet: Gene expression analysis of lymphocyte activation related transcription factors in Jurkat transiently RIZ overexpressing cells. PRDM1 transcript levels were compared in Jurkat cells transiently transfected with the indicated encoding plasmids (with the expression value of the empty vector equal to 1). The ratio between the PR- and PR + transcripts for each gene was also calculated. RIZ2 overexpression reduced the ratio between BLIMP1a and BLIMP1b in favor of BLIMP1b . The expression of GATA3 , TBX21 , FOXP3 , RORC , CD40LG was also measured in Jurkat cells in both basal conditions (ΔCt method as fold on RPS18 control gene) and after 36 h transient transfection with the plasmid encoding for RIZ1, RIZ2 or with pSG5 control (ΔΔCt method with the indicated control gene). Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Western blot analysis was performed as described elsewhere [ ] with mouse monoclonal antibody FLAGTM clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).

Techniques: Gene Expression, Activation Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Control

Journal: Journal of Translational Medicine

Article Title: Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

doi: 10.1186/s12967-023-04066-x

Figure Lengend Snippet: A Gene expression analysis of the main transcription factors related to lymphocyte activation in Jurkat stably transfected cells. qRT-PCR analysis of BLIMP1a , BLIMP1b , GATA3 , FOXP3 , RORC , TBX21 , BCL6 , CD40LG , KLF2 , CTLA4 , IL2 , IL2RA , CCR4 , CCR6 , and CXCR3 on Jurkat cells transfected with the plasmid encoding for RIZ1, RIZ2 or with pSG5 control vector and treated with or without treatment with polyclonal activators for 6 h. Three independent experiments in triplicates were performed and data expressed as mean ± SD. ns (not significant), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: Western blot analysis was performed as described elsewhere [ ] with mouse monoclonal antibody FLAGTM clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).

Techniques: Gene Expression, Activation Assay, Stable Transfection, Transfection, Quantitative RT-PCR, Plasmid Preparation, Control

Journal: Journal of Translational Medicine

Article Title: Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

doi: 10.1186/s12967-023-04066-x

Figure Lengend Snippet: Expression correlation analysis of PRDM1 , PRDM2 and the main transcripts with the indicated gene/transcript was performed on GEPIA2 platform using the Spearman correlation coefficient R. Datasets evaluated: whole blood, thymoma normal and EBV-transformed lymphocytes. PRDM2-001 from GEPIA2 database represents RIZ1 transcript, PRDM2-003 represents RIZ2 transcript, PRDM1-001 represents BLIMP1a transcript and PRDM1-002 represents BLIMP1b transcript. Scatter plots of mRNA levels were normalized by RPS18 expression. A Correlation analysis between PRDM1 and PRDM2 gene expression; B Correlation between PRDM1, PRDM2 and their main transcripts with GATA3 ; C Correlation between PRDM1 , PRDM2 and their main transcripts with the indicated signatures

Article Snippet: Western blot analysis was performed as described elsewhere [ ] with mouse monoclonal antibody FLAGTM clone M2 (Sigma-Aldrich) and rabbit polyclonal antibody RIZ1 (Abcam Ltd.).

Techniques: Expressing, Transformation Assay, Gene Expression

Journal: Journal of Hematology & Oncology

Article Title: RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression

doi: 10.1186/1756-8722-2-28

Figure Lengend Snippet: RIZ1 expression in bone marrow of CML patients . ( a ) Immunohistochemical analysis of matched bone marrow trephine biopsies and bone marrow aspirate clot samples from patients in chronic phase and accelerated phase or myeloid blast crisis using an anti-RIZ1 antibody. ( b ) RIZ1 expression in normal bone marrow and normal bone marrow staining in the absence of RIZ1 primary antibody (Negative control). ( c ) Immunohistochemical analysis of RIZ1 expression in unmatched patient bone marrow biopsies and clot sections from chronic phase (CP) (N = 10), accelerated phase (AP) (N = 7) and blast crisis (BC) (N = 15) using an anti-RIZ1 monoclonal antibody. Relative RIZ1 expression represents 3,3-diaminobenzidine chromagen intensity. Mean RIZ1 expression for each group is shown as a black line and errors bars represent the standard deviation.

Article Snippet: Immunohistochemical analysis of B5 fixed/paraffin embedded and decalcified bone marrow trephine biopsies and B5 fixed/paraffin embedded bone marrow aspirate clot samples was performed using an anti-RIZ1 monoclonal antibody (Abgent, San Diego, CA, USA) (1:25 dilution) and a horseradish peroxidase-coupled secondary antibody.

Techniques: Expressing, Immunohistochemical staining, Staining, Negative Control, Standard Deviation

Journal: Journal of Hematology & Oncology

Article Title: RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression

doi: 10.1186/1756-8722-2-28

Figure Lengend Snippet: RIZ1 expression in G-CSF mobilized peripheral blood . Flow cytometry analysis of RIZ1 protein expression in granulocytes, monocytes, and CD34 + cells. (Con) represents flow cytometry analysis in the absence of the RIZ1 primary antibody.

Article Snippet: Immunohistochemical analysis of B5 fixed/paraffin embedded and decalcified bone marrow trephine biopsies and B5 fixed/paraffin embedded bone marrow aspirate clot samples was performed using an anti-RIZ1 monoclonal antibody (Abgent, San Diego, CA, USA) (1:25 dilution) and a horseradish peroxidase-coupled secondary antibody.

Techniques: Expressing, Flow Cytometry

Journal: Journal of Hematology & Oncology

Article Title: RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression

doi: 10.1186/1756-8722-2-28

Figure Lengend Snippet: Effect of RIZ1 expression on cell viability and apoptosis in CML myeloid blast crisis model cell lines . ( a ) Viability assay for cell lines transfected with pRIZ1 (dashed line) or pcDNA3 control plasmid (solid line). ( b ) Annexin V assay of ERY-1, YN-1, JURL-MK1, and K562 one day after transfection with pRIZ1 (+) or pcDNA3 control plasmid (-). Percentages of apoptotic cells were detected using annexin V-FITC and PI staining. Total percentage of cells undergoing early and end stage apoptosis are indicated. White histogram represents cells in early apoptosis (FITC + , PI - ). Black histogram represents cells that are in the end stage of apoptosis or that are already dead (FITC + , PI + ). Error bars represent standard deviation from three independent experiments.

Article Snippet: Immunohistochemical analysis of B5 fixed/paraffin embedded and decalcified bone marrow trephine biopsies and B5 fixed/paraffin embedded bone marrow aspirate clot samples was performed using an anti-RIZ1 monoclonal antibody (Abgent, San Diego, CA, USA) (1:25 dilution) and a horseradish peroxidase-coupled secondary antibody.

Techniques: Expressing, Viability Assay, Transfection, Plasmid Preparation, Annexin V Assay, Staining, Standard Deviation

Journal: Journal of Hematology & Oncology

Article Title: RIZ1 is potential CML tumor suppressor that is down-regulated during disease progression

doi: 10.1186/1756-8722-2-28

Figure Lengend Snippet: Effect of RIZ1 expression on differentiation in CML myeloid blast crisis model cell lines . ( a ) Benzidine staining assays comparing erythroid differentiation in K562 cells transfected with shRNA non-silencing control plasmid (K562), K562+RIZ1 cells transfected with shRNA non-silencing control plasmid (K562+RIZ1), and K562+RIZ1 cells transfected with pRIZ1shRNA (K562+RIZ1+shRNA). ( b ) Western analysis of RIZ1 expression in K562 transfected with shRNA non-silencing control plasmid (K562), K562+RIZ1 cells transfected with shRNA non-silencing control plasmid (K562+RIZ1), and K562+RIZ1 cells transfected with pRIZ1shRNA (K562+RIZ1+shRNA). ( c ) RT-PCR analysis of RIZ1 mRNA expression in ERY-1 and YN-1 transfected with shRNA non-silencing control plasmid (Con shRNA) or with pRIZ1shRNA (RIZ1 shRNA). Total RNA was reverse transcribed and cDNA amplified with RIZ1 and β-actin-specific primers. M represent DNA ladder and H2O represents RT-PCR reaction without template DNA. ( d ) Erythroid differentiation assay comparing ERY-1 and YN-1 after transfection with pRIZ1shRNA or shRNA non-silencing control plasmid (Con). Cell lines were transfected with pRIZ1shRNA or shRNA non-silencing control plasmid and cultured for three days. Histograms show the percentage of benzidine-positive cells that were scored by light microscopy. Error bars represent the standard deviation from three independent experiments. ( e ) CD33 and CD117 expression in JURL-MK1 cells as compared with JURL-MK1 cells expressing RIZ1 (JURL-MK1+pRIZ). JURL-MK1 was transfected with pRIZ1 or pcDNA3 control plasmid (con) and cultured for three days. Panel ( i ) shows the fluorescence intensity of phycoerythrin (PE)-conjugated antibody against CD33. Panel ( ii ) shows the fluorescence intensity of (PE)-conjugated antibody against CD117. Panels ( iii ) and ( iv ) show immunocytochemical staining using an anti-CD117 antibody in JURL-MK and JURL-MK1+pRIZ1 cells, respectively.

Article Snippet: Immunohistochemical analysis of B5 fixed/paraffin embedded and decalcified bone marrow trephine biopsies and B5 fixed/paraffin embedded bone marrow aspirate clot samples was performed using an anti-RIZ1 monoclonal antibody (Abgent, San Diego, CA, USA) (1:25 dilution) and a horseradish peroxidase-coupled secondary antibody.

Techniques: Expressing, Staining, Transfection, shRNA, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Differentiation Assay, Cell Culture, Light Microscopy, Standard Deviation, Fluorescence

Journal: Frontiers in Oncology

Article Title: Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients

doi: 10.3389/fonc.2019.01396

Figure Lengend Snippet: RIZ1 expression was downregulated in cervical cancer tissues compared with the adjacent non-tumor tissues. (A) Representative photomicrographs of RIZ1 immunohistochemical staining. Panel 1, positive RIZ1 expression in cervical squamous cell carcinoma; panel 2, positive RIZ1 expression in cervical cervical adenocarcinoma; panel 3, positive RIZ1 expression in normal cervical tissue; panel 4, negative RIZ1 expression in cervical squamous cell carcinoma; panel 5, negative RIZ1 expression in cervical cervical adenocarcinoma; panel 6, negative RIZ1 expression in normal cervical tissue. Original magnification, ×200. (B) Total proteins from four pairs of cervical cancer tissues (T) and the adjacent non-tumor tissues (N) were extracted for immunoblotting of RIZ1 with β-actin as a loading control. (C) Relative expression RIZ1 to β-actin was quantitated by ImageJ. (D) Cervical cancer tissues (T) and the adjacent non-tumor tissues (N) were extracted for quantitative real-time RT-PCR analysis of RIZ1 mRNA with β-actin as an internal control.

Article Snippet: The primary anti-RIZ1 antibody (cat. AM1194A, Abgent, San Diego, CA, USA) was used at a dilution of 1:50.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients

doi: 10.3389/fonc.2019.01396

Figure Lengend Snippet: Association analysis between the expression level of RIZ1 and the clinicopathologic factors of FIGO stage I and II cervical cancer patients.

Article Snippet: The primary anti-RIZ1 antibody (cat. AM1194A, Abgent, San Diego, CA, USA) was used at a dilution of 1:50.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients

doi: 10.3389/fonc.2019.01396

Figure Lengend Snippet: Low RIZ1 expression predicted shorter overall survival (OS) and disease-free survival (DFS) time of cervical cancer patients. Kaplan–Meier curves for the survival of prognosis in 268 patients with FIGO stages I–II cervical cancer according to the categories of negative and positive RIZ1 expression (analyzed with log-rank test). (A) Overall survival; (B) disease-free survival.

Article Snippet: The primary anti-RIZ1 antibody (cat. AM1194A, Abgent, San Diego, CA, USA) was used at a dilution of 1:50.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients

doi: 10.3389/fonc.2019.01396

Figure Lengend Snippet: Overexpression of RIZ1 inhibited the proliferation of cervical cancer cells. (A) HeLa cells and SiHa cells were transfected with plasmid pcDNA3.1-RIZ1-AE or the empty vector pcDNA3.1. 48 h post-transfection, total proteins from each cell lines were extracted for immunoblotting of RIZ1 with β-actin as a loading control. (B) Relative expression RIZ1 to β-actin was quantitated by ImageJ. (C) HeLa cells were transfected with plasmid pcDNA3.1-RIZ1-AE or the empty vector pcDNA3.1. The cell viability on day 1 to day 5 after transfection was assessed by MTT assay. (D) SeHa cells were transfected with plasmid pcDNA3.1-RIZ1-AE or the empty vector pcDNA3.1. The cell viability on day 1 to day 5 after transfection was assessed by MTT assay. Each point indicates the mean of spectrometric absorbance ± SD of three independent experiments. * P < 0.05, ** P < 0.005.

Article Snippet: The primary anti-RIZ1 antibody (cat. AM1194A, Abgent, San Diego, CA, USA) was used at a dilution of 1:50.

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Expressing, MTT Assay

Journal: Frontiers in Oncology

Article Title: Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients

doi: 10.3389/fonc.2019.01396

Figure Lengend Snippet: RIZ1 overexpression induced cell cycle arrest at the G2-M phase and apoptosis of cervical cancer cells. HeLa cells and SiHa cells were transfected with plasmid pcDNA3.1-RIZ1-AE or the empty vector pcDNA3.1 for 48 h. (A) Cell cycle distribution was assessed with PI staining followed by flow cytometry. (B) Percentage of cells at different phases, (C) Cell apoptosis was assessed with Annvin V staining followed by flow cytometry, (D) Percentage of Annexin V positive cells. The data are the mean ± SD of one representative experiment. Similar results were obtained in three independent experiments.

Article Snippet: The primary anti-RIZ1 antibody (cat. AM1194A, Abgent, San Diego, CA, USA) was used at a dilution of 1:50.

Techniques: Over Expression, Transfection, Plasmid Preparation, Staining, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: Decreased Expression of Retinoblastoma Protein-Interacting Zinc-Finger Gene 1 Is Correlated With Poor Survival and Aggressiveness of Cervical Cancer Patients

doi: 10.3389/fonc.2019.01396

Figure Lengend Snippet: RIZ1 inhibited the migration and invasion of cervical cancer cells in vitro . (A,B) RIZ1 overexpression reduced the migration capacity of HeLa and SiHa cells as determined by a wound-healing assay, ** P < 0.01. (C,D) RIZ1 overexpression reduced the invasion capacity of HeLa and SiHa cells was determined by Transwell assays, ** P < 0.01.

Article Snippet: The primary anti-RIZ1 antibody (cat. AM1194A, Abgent, San Diego, CA, USA) was used at a dilution of 1:50.

Techniques: Migration, In Vitro, Over Expression, Wound Healing Assay