Journal: Frontiers in Molecular Neuroscience

Article Title: Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene ( Plaur ) and Its Molecular Target Mef2d

doi: 10.3389/fnmol.2022.865858

Figure Lengend Snippet: Analysis of Plaur-miR1-3p and Plaur-miR1-5p expression in Neuro2a cells and mouse posterior cortex. (A) qPCR of Plaur-miR1-3p and Plaur-miR1-5p expression in Neuro2a cells. The data are expressed as mean ± SEM ( n = 3), normalized to Snord95 expression as a reference gene. (B) qPCR of Plaur expression in Neuro2a cells. The data are expressed as mean ± SEM ( n = 3), normalized to Actb (encodes β-actin) expression as a reference gene. For (A,B) , the columns are: Neuro2a WT—control Neuro2a cells; Neuro2a-KO-uPAR—uPAR-deficient Neuro2a cells; Neuro2a Plaur-miR1—Neuro2a cells with ectopic Plaur-pre-miR1 expression; Neuro2a-KO-uPAR Plaur-miR1—uPAR-deficient Neuro2a cells with ectopic Plaur-pre-miR1 expression. The data were analyzed by using analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism software. (C) qPCR of Plaur-miR1-5p and Plaur-miR1-3p in the posterior cortex 0 and 3 h after endogenous Plaur induction. The data are expressed as mean ± SEM ( n = 4). The data were analyzed by using a one-sample t -test with GraphPad Prism software. Statistical significance in (A–C) is indicated by bars and asterisks as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Sequence results of 19 TA vector clones containing Plaur-miR1-5p sequences generated in (C) . Four of 19 (20%) clones were 22–24 bp in length and could correspond to mature miRNA. In addition, 8 of 19 (40%) clones were 22–31 bp in length and could correspond to other RNA fragments isolated from posterior cortex small RNA fraction . (E) Clones 1, 9, 11, and 16 show the Plaur-miR1-5p sequence, demonstrated as alignment with Plaur-pre-miR1. The asterisks in (D,E) indicate that the aligned sequences match at that position.

Article Snippet: Mouse Neuro2a neuroblastoma cells (ATCC ® CCL-131TM University Boulevard Manassas, VA, United States) not exceeding 20 passages were cultured in complete medium—Dulbecco’s Modified Eagle’s Medium (DMEM) (#21969035), 10% fetal bovine serum (FBS, Gibco, #10270-106, United Kingdom), 1 × Minimum Essential Medium (MEM) Non-Essential Amino Acids Solution (#11140050) and 1 × antibiotic-antimycotic solution (#15240062; all from Gibco, Life Technologies, Bleiswijk, Netherlands)—at 37 ° C in an atmosphere with 5% CO 2 .

Techniques: Expressing, Control, Software, Sequencing, Plasmid Preparation, Clone Assay, Generated, Isolation

Journal: Frontiers in Molecular Neuroscience

Article Title: Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene ( Plaur ) and Its Molecular Target Mef2d

doi: 10.3389/fnmol.2022.865858

Figure Lengend Snippet: Plaur-miR1 (Plaur-miR1-3p and Plaur-miR1-5p) expression altered the expression of the predicted target genes Mef2d and Snrnp200 in Neuro2a cells. qPCR analysis of (A) Mef2d and (B) Snrnp200 in Neuro2a WT, Neuro2a KO uPAR, Neuro2a WT, and Neuro2a KO uPAR cells after pBl-U6-Plaur-pre-miR1 expression. The data presented as mean ± SEM ( n = 3), normalized to Actb (encodes β-actin) expression as a reference gene. The data were analyzed by using analysis of variance followed by Dunnett’s multiple comparisons test with GraphPad Prism software. Statistical significance is indicated by bars and asterisks as follows: ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse Neuro2a neuroblastoma cells (ATCC ® CCL-131TM University Boulevard Manassas, VA, United States) not exceeding 20 passages were cultured in complete medium—Dulbecco’s Modified Eagle’s Medium (DMEM) (#21969035), 10% fetal bovine serum (FBS, Gibco, #10270-106, United Kingdom), 1 × Minimum Essential Medium (MEM) Non-Essential Amino Acids Solution (#11140050) and 1 × antibiotic-antimycotic solution (#15240062; all from Gibco, Life Technologies, Bleiswijk, Netherlands)—at 37 ° C in an atmosphere with 5% CO 2 .

Techniques: Expressing, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene ( Plaur ) and Its Molecular Target Mef2d

doi: 10.3389/fnmol.2022.865858

Figure Lengend Snippet: Results of the luciferase reporter assay for the confirmation of specificity of Plaur-miR1-5p effect on its predicted targets. Neuro2a cells were co-transfected with pBl-U6-Plaur-pre-miR1 and pGL3 vector coding 3-UTR or CDS sequences. Empty vectors pBl-U6 and pGL3 were used as controls. Data were normalized by luciferase activity in Neuro2a cells co-transfected with pBl-U6-Plaur-pre-miR1 and pGL3 vectors. The data are presented as mean ± SEM ( n = 3) and compared using two-way ANOVA followed by Šídák’s multiple comparisons test with GraphPad Prism software. Statistical significance is indicated by bars and asterisks as follows: * p < 0.05.

Article Snippet: Mouse Neuro2a neuroblastoma cells (ATCC ® CCL-131TM University Boulevard Manassas, VA, United States) not exceeding 20 passages were cultured in complete medium—Dulbecco’s Modified Eagle’s Medium (DMEM) (#21969035), 10% fetal bovine serum (FBS, Gibco, #10270-106, United Kingdom), 1 × Minimum Essential Medium (MEM) Non-Essential Amino Acids Solution (#11140050) and 1 × antibiotic-antimycotic solution (#15240062; all from Gibco, Life Technologies, Bleiswijk, Netherlands)—at 37 ° C in an atmosphere with 5% CO 2 .

Techniques: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Software

Journal: Cell Reports Medicine

Article Title: CD103 + CD8 + T RM Cells Accumulate in Tumors of Anti-PD-1-Responder Lung Cancer Patients and Are Tumor-Reactive Lymphocytes Enriched with Tc17

doi: 10.1016/j.xcrm.2020.100127

Figure Lengend Snippet:

Article Snippet: anti-human pSTAT3pS727-APC (REA324) , Miltenyi Biotec , Cat# 130-105-000; RRID: AB_2653576.

Techniques: Purification, Recombinant, Isolation, Staining, RNA Library Preparation, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software