Journal: RNA

Article Title: 3′ Uridylation controls mature microRNA turnover during CD4 T-cell activation

doi: 10.1261/rna.060095.116

Figure Lengend Snippet: TUT4 and TUT7 expression is down-regulated after T-cell activation: ( A ) RNA levels of MTPAP, GLD2, PAPD5, TUT4, POLS, and TUT7 were assessed by RT-qPCR in freshly isolated mouse naïve CD4 T cells or cells activated with anti-CD3 and anti-CD28 for 48 h. mRNA levels were normalized to β-actin and Yhwaz housekeeping genes and are presented in arbitrary units ( n = 7). ( B ) TUT4 mRNA levels in human T lymphoblasts measured by RT-qPCR after two different activation stimuli: phorbol myristate acetate (PMA) with ionomycin and anti-CD3 plus anti-CD28 ( n = 3). ( C ) Mouse naïve CD4 T cells from OT-II mice specific to ovalbumin peptide were co-cultured with dendritic cells in the absence or presence of the peptide. TUT4 mRNA levels were determined by RT-qPCR as in A ( n = 5). ( D ) Long-term time course of TUT4 mRNA levels in activated mouse CD4 T cells ( n = 3). ( E ) Western blot analysis of TUT4 protein content in CD4 T cells after activation with anti-CD3 plus anti-CD28 or with concanavalin A (ConA) followed by expansion with interleukin 2 (IL-2). Representative immunoblots ( n = 3). ERMs were included as a loading control. ( F ) Western blot analysis of TUT7 protein at different time points after antibody activation of CD4 T cells. Representative immunoblots ( n = 3). p150 was included as a loading control. Numbers below blots show normalized densitometry values relative to naïve T cells. Error bars in A – C represent standard deviation; (***) P < 0.001; (**) P < 0.05; ns, nonsignificant.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Zcchc11 (TUT4), rabbit polyclonal anti-Zcchc6 (TUT7) (Proteintech), goat polyclonal anit-Zcchc11 (ProSci), anti-p150glued (BD Transduction Laboratories), monoclonal anti-EZH2 (Cell Signaling), polyclonal anti ezrin/moesin (ERMs) (90/3) (provided by Heinz Furthmayr, Stanford University, Stanford, CA).

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Isolation, Cell Culture, Western Blot, Control, Standard Deviation

Journal: RNA

Article Title: 3′ Uridylation controls mature microRNA turnover during CD4 T-cell activation

doi: 10.1261/rna.060095.116

Figure Lengend Snippet: TUT4-dependent uridylation of mature microRNA. Small RNAs from naïve wild-type or TUT4-deficient CD4 T cells were analyzed by deep sequencing. ( A – D ) Fold changes between wild-type and TUT4−/− CD4 T cells calculated from oligo- ( A ) and mono-uridylated miRNAs ( B ) as well as mono- ( C ) and oligo-adenylated miRNA counts ( D ). Only modified miRNAs whose expression level is above log 2 > 2 are presented. A negative fold change indicates lower levels in TUT4-deficient compared to wild-type. ( E ) Comparison of the levels of the individual canonical miRNAs corresponding to the identified TUT4 uridylation targets between wild-type and TUT4-deficient CD4 T cells. ( F ) Comparison of the levels of individual uridylated miRNAs identified as TUT4 targets between naïve and activated wild-type CD4 T cells.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Zcchc11 (TUT4), rabbit polyclonal anti-Zcchc6 (TUT7) (Proteintech), goat polyclonal anit-Zcchc11 (ProSci), anti-p150glued (BD Transduction Laboratories), monoclonal anti-EZH2 (Cell Signaling), polyclonal anti ezrin/moesin (ERMs) (90/3) (provided by Heinz Furthmayr, Stanford University, Stanford, CA).

Techniques: Sequencing, Modification, Expressing, Comparison

Journal: RNA

Article Title: 3′ Uridylation controls mature microRNA turnover during CD4 T-cell activation

doi: 10.1261/rna.060095.116

Figure Lengend Snippet: Uridylation of mature miRNAs determines their turnover during T-cell activation. ( A ) Diagram representing the qPCR method used to distinguish between di-uridylated synthetic miRNAs and unmodified ones. After a linker ligation and reverse transcription, custom probes specific for the 3′ end were used in combination with reverse and forward primers for the miRNA. ( B ) Synthetic miRNAs were nucleofected into naïve CD4 T cells, which were then activated with anti-CD3 and anti-CD28 or left resting in the presence of IL-7. Levels of the synthetic miRNAs were analyzed by a variation of MiQPCR. Synthetic RNA levels were normalized to endogenous controls snoRNA 420 and snoRNA 412 and are presented in arbitrary units ( n = 3). ( C ) Scheme of the proposed mechanism of miRNA post-transcriptional regulation during T-cell activation. Naïve cells under steady-state contain basal levels of uridylated miRNAs that are down-regulated after T-cell activation. This decrease is accompanied by the down-regulation of uridyltransferases TUT4 and TUT7. Additionally, we hypothesize that a putative exonuclease might be induced in activated T cells responsible for uridylated miRNAs degradation.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-Zcchc11 (TUT4), rabbit polyclonal anti-Zcchc6 (TUT7) (Proteintech), goat polyclonal anit-Zcchc11 (ProSci), anti-p150glued (BD Transduction Laboratories), monoclonal anti-EZH2 (Cell Signaling), polyclonal anti ezrin/moesin (ERMs) (90/3) (provided by Heinz Furthmayr, Stanford University, Stanford, CA).

Techniques: Activation Assay, Ligation, Reverse Transcription

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Expression of TMEM230 in brain Glioblastoma Multiforme (GBM) and Low-Grade Gliomas (LGG) analyzed from The Cancer Genome Atlas. (A) Glioblastoma multiforme tumors showed significantly elevated level of TMEM230 mRNA compared to low-grade gliomas (unpaired t -test p < 0.0001). Low-grade gliomas consist of astrocytoma, oligoastrocytoma and oligodendroglioma patient samples. (B) Poor prognosis was correlated with high TMEM230 in low-grade gliomas. (C) Poor prognosis was correlated with high TMEM230 in astrocytoma (top), oligoastrocytoma (middle) and oligodendroglioma (bottom). Relationship between TMEM230 expression levels and prognosis of low-grade gliomas affected patients indicated that lower expression of TMEM230 was associated with increased overall survival. Each glioma subtype is indicated by the median of gene expression of TMEM230 . B and C analyses were generated from the Kaplan-Meier test based on the expression of medium TMEM230. (D) Representative heatmap displaying the most variable expressed genes for astrocytoma using the R with “pheatmap” package for which functional enrichment was generated with DAVID program.

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Expressing, Gene Expression, Generated, Functional Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Validation of endogenous and lentivirus downregulation of TMEM230 mRNA and protein expression in U87-MG cells. (A) Validation of constitutive downregulation of endogenous TMEM230 transcript expression with lentiviral system. Endogenous control: HPRT, Error bars represent 95% confidence interval. (B) Validation of constitutive downregulation of endogenous TMEM230 protein expression with the lentiviral system. Endogenous control: β-actin. (C) Western blot analysis (top panel) showed TMEM230 protein was not detected in fetal bovine serum (FBS), serum replacement (SR) or conditioned media (CM) containing serum replacement or FBS (CM SR and CM FBS) obtained from endogenous TMEM230 expressing U87 cells. Ponceau S staining (lower panel) showed an abundance of protein was loaded. (D) Coomassie blue staining showed an increase of expression of extracellular vesicle membrane protein, CD81 in conditioned media of U87 cells constitutively over-expressing TMEM230 with respect to conditioned media collected from control cells (U87GFP and U87shSCR). Detection of CD81 (D) but lack of detection of TMEM230 protein in culture media (C) supports that the TMEM230 protein regulates extracellular vesicle generation or secretion but is not itself a component of extracellular vesicles.

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Biomarker Discovery, Expressing, Control, Western Blot, Staining, Membrane

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Downregulation of TMEM230 was sufficient to promote loss of U87-MG substratum adhesion capacity in fetal bovine serum containing media. Control (U87shSCR) and U87 cells in which TMEM230 was constitutively downregulated (U87shTMEM230) were cultured in extracellular vesicle depleted FBS (Fetal Bovine Serum) containing culture media. Equal number of control cells and cells in which endogenous TMEM230 was downregulated were plated (P0) in vesicle depleted FBS containing culture media and monitored over 72 h, starting from when green fluorescent protein (GFP) expression was first observed (0 h, not shown). Cells in which TMEM230 were downregulated displayed decrease in cytoplasm dimensions and disrupted cytoplasmic invadopodium like extensions. Cells were re-passaged (P1) and monitored for additional 144 h. Re-passaged cells (P1) displayed a more limited capacity for cell adhesion, supporting that TMEM230 is necessary for scaffold attachment of cells.

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Control, Cell Culture, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Downregulation of endogenous TMEM230 was sufficient to promote loss of U87-MG substratum adhesion capacity in serum replacement containing media. Control (U87shSCR) and U87 cells in which TMEM230 was constitutively downregulated (U87shTMEM230) were cultured in serum replacement (SR) containing media (P0). Equal number of control cells and cells in which TMEM230 was downregulated were plated in SR containing culture media and monitored over 192 h, starting from when GFP expression was first observed (0 h).

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Control, Cell Culture, Expressing

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Human umbilical vein endothelial cells cultured in FBS and serum replacement containing conditioned media from U87-MG cells expressing endogenous TMEM230 promoted angiogenic behavior. (1–4) Representative images at 24 h of human umbilical vein endothelial cells (HUVEC) in Matrigel treated with conditioned media obtained from 3 days cultures of U87 control (U87shSCR) and U87 in which TMEM230 was down regulated (U87shTMEM230). (5–15) Human umbilical vein endothelial cells in 3D treated with conditioned media obtained from 3 days cultures of U87 control and U87 which TMEM230 was down regulated shown for without (5–9) and with angiogenic factors (11–14) . U87 cells were cultured in media containing fetal bovine serum (FBS) or serum replacement (SR).

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Cell Culture, Expressing, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Endogenous TMEM230 promoted U87-MG cell migration, tumor-endothelial cell contact and displacement in co-culture assays. (A) Low magnification shows the co-culture assay set up. (B) Representative images showing the periphery (outgrowth) and core (initial location of cell plating) of U87shTMEM230 cells at 48 h. Downregulation of TMEM230 in U87 cells was associated with cytoplasm of reduced mass, disrupted cytoplasmic invadopodium like extensions, decreased cell anchorage and reduced contacts among initially confluent plated cells. (C) Representative images of U87shTMEM230 cells at periphery and core at 9 days. U87 cells with reduced TMEM230 expression displayed reduced anchorage capacity and motility (see red circle at periphery of initial site of plating of the confluent cells) compared to control cells. (D–F) Displacement of the confluent human umbilical vein endothelial cells by U87 control cells (U87shSCR) expressing endogenous TMEM230 through infiltration into the confluent mass of human umbilical vein endothelial cells (see red circles), a behavior that is associated with the first step of intussusceptive induced blood vessel branching.

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Migration, Co-Culture Assay, Co-culture Assay, Expressing, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: Endogenous expression of TMEM230 promoted U87-MG migration and tubule like structure formation recapitulating a vascular mimicry like behavior. (A) Representative 3D bodies or structures (only the borders of a larger 3D body of U87shSCR or a complete body of U87shTMEM230 cells are shown by red circles) of U87 cells expressing endogenous TMEM230 displayed vascular mimicry, cell sprouting, collective cell movement, and invasion in 3D Matrigel. U87 cells in which endogenous TMEM230 was downregulated did not generate 3D bodies of significant size in agreement that TMEM230 was required for U87 cell growth. Two different media were used for generating VM like structures from U87 cells, media used for culturing adherent U87 tumor cells or HUVEC shown in , (top panel), respectively. (B) Higher magnification of control cells.

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Expressing, Migration, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy

doi: 10.3389/fncel.2021.703431

Figure Lengend Snippet: All enriched pathways identified with high or low TMEM230 expression in diverse patient glioma tumors. (A) Enriched pathways identified common between high or low TMEM230 expression in all LGG of patient. (B) Scheme for identifying different pathways between high or low TMEM230 expression in LGG and glioblastomas. (C) Enriched pathways identified different between high or low TMEM230 expression in LGG and glioblastoma. See corresponding .

Article Snippet: Antibodies used were polyclonal rabbit anit-C20ORF30 (TMEM230, 1:1,000, Santa Cruz, sc-85410), monoclonal mouse anti-MOESIN (1:200, Santa Cruz, sc-58806), polyclonal rabbit anti-CD138 (Syndecan-1, 1:250, Thermo Fisher Scientific, 36-2900), anti-HCAM (CD44, 1:1,000, Santa Cruz, sc-9960) and polyclonal goat anit-β-ACTIN (1:2,000, Santa Cruz, sc-1615, used as endogenous control).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Mechanism of Paeoniflorin on ANIT-Induced Cholestatic Liver Injury Using Integrated Metabolomics and Network Pharmacology

doi: 10.3389/fphar.2021.737630

Figure Lengend Snippet: Effect of PF on the expression of CYP2C9, ABCB1, MAOB, CDC25B, and MTOR in the treatment of cholestatic liver injury detected by western blotting. (A) Western blotting images of CYP2C9, ABCB1, MAOB, CDC25B, and MTOR; (B) Relative protein expression of CDC25B; (C) Relative protein expression of CYP2C9; (D) Relative protein expression of MAOB; (E) Relative protein expression of ABCB1; (F) Relative protein expression of MTOR. The data are expressed as the mean ± SD, n = 3. ## p < 0.01, # p < 0.05 compared with the control group, ** p < 0.01, * p < 0.05 compared with ANIT group.

Article Snippet: The PVDF membranes were blocked with 5% fat-free milk at room temperature for 2 hours, then incubated overnight at 4°C with antibodies against anit-CDC25B (PB9488, BOSTER, dilution: 1:1,000), anit-MTOR (A00003-2, BOSTER, dilution: 1:1,000), anit-CYP2C9 (16546-1-AP, proteintech, dilution: 1:2,000), anit-ABCB1 (A00049-3, BOSTER, dilution: 1:1,000), anit-MAOB (PB9665, BOSTER, dilution: 1:1,000).

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Mechanism of Paeoniflorin on ANIT-Induced Cholestatic Liver Injury Using Integrated Metabolomics and Network Pharmacology

doi: 10.3389/fphar.2021.737630

Figure Lengend Snippet: Effect of PF on the expression of CYP2C9, ABCB1, MAOB, CDC25B, and MTOR in the treatment of cholestatic liver injury. (A) The expression of CYP2C9 in the liver tissue; (B) The expression of ABCB1 in the liver tissue; (C) The expression of MAOB in the liver tissue; (D) The expression of CDC25B in the liver tissue; (E) The expression of MTOR in the liver tissue. The data are expressed as the mean ± SD, n = 3. ## p < 0.01, # p < 0.05 compared with the control group, ** p < 0.01, * p < 0.05 compared with ANIT group.

Article Snippet: The PVDF membranes were blocked with 5% fat-free milk at room temperature for 2 hours, then incubated overnight at 4°C with antibodies against anit-CDC25B (PB9488, BOSTER, dilution: 1:1,000), anit-MTOR (A00003-2, BOSTER, dilution: 1:1,000), anit-CYP2C9 (16546-1-AP, proteintech, dilution: 1:2,000), anit-ABCB1 (A00049-3, BOSTER, dilution: 1:1,000), anit-MAOB (PB9665, BOSTER, dilution: 1:1,000).

Techniques: Expressing