flag p50  (Addgene inc)

Journal: Research Square

Article Title: Transgelin 2 guards T cell lipid metabolic programming and anti-tumor function

doi: 10.21203/rs.3.rs-3683989/v1

Figure Lengend Snippet: a , Pre-activated CD8 + T cells from WT C57BL/6J mice were treated with 2-Deoxy-D-glucose (2-DG), Tunicamycin (TM) or Thapsigargin (TG) at the indicated concentrations. Xbp1s and Tagln2 expression were determined via qRT-PCR 16 h after 2-DG or TM treatment, and 6 h post TG exposure. Data were normalized to Actb in each sample ( n = 3 per condition). b , TAGLN2 protein expression levels determined by FACS ( n = 3 per group) in the same samples described in ( a ). c , Representative confocal images for TAGLN2 expression in CD8 + T cells from WT C57BL/6J mice under the indicated conditions from two independent experiments. d-g , Naïve CD8 + T cells isolated from Ern1 fl/fl or Ern1 fl/fl Cd4 Cre mice ( d,e ) or Xbp1 fl/fl or Xbp1 fl/fl Cd4 Cre mice ( f,g ) were cultured under the indicated conditions. Expression of the Tagln2 transcript was determined by RT-qPCR, and data were normalized to endogenous levels of Actb in each sample ( d,f ) ( n = 3 per condition and genotype). Representative FACS histograms and quantitative analysis of TAGLN2 protein levels in CD8 + CD44 + T cells under the indicated conditions ( e,g ) ( n = 3 per genotype). h , Pre-activated CD8 + T cells from WT C57BL/6J mice were stimulated via CD3/CD28 for 16 h in the absence or presence of TM (1 μg/ml). White bars, DMSO; Red bars, MKC8866 (2 μM); Orange bars, KIRA8 (1 μM). Expression of Xbp1s and Tagln2 transcripts were determined by RT-qPCR, and data were normalized to endogenous levels of Actb in each sample ( n = 3 per condition). i , Conserved XBP1s-binding motifs (CACGTC) from mouse (top) and human (bottom) are shown. j , Pre-activated CD8 + T cells from WT C57BL/6J mice were stimulated via CD3/CD28 for 16 h in the absence or presence of the ER stressor TM (1 μg/ml). ChIP assays were performed using anti-XBP1s or isotype control antibodies. qRT-PCR was used to determine XBP1s occupancy at two XBP1s-binding sites (BS1 and BS2) in Tagln2 promoter regions under the conditions tested. No XBP1s binding region (NR) was used as a negative control. ChIP-quantitative PCR assays were performed using T cells from three independent mice ( n = 3 per condition). k , Tagln2 promoter-luciferase construct (−738 to +134) was co-transfected with the combination of NF-kB (p50) and XBP1s expressing vectors in HEK-293 T cells. Lysates were prepared 48 h after transfection, and luciferase activities were measured with the firefly luciferase activities normalized to renilla luciferase activities ( n = 3). l,m , Representative FACS histograms and quantitative analysis of TAGLN2 protein levels in effector (CD62L low CD44 high ) or central memory (CD62L high CD44 high ) CD8 + intratumoral T cells in omentum ( l ) and solid tumor ( m ) from female mice of the indicated genotypes bearing PPNM-based HGSC for 40 days ( Xbp1 fl/fl , n = 5; Xbp1 fl/fl Cd4 Cre , n = 8). n , UMAP plot of T cell subtypes from 11 HGSOC treatment-naïve human tumor specimens. o , GSEA enrichment plots showing downregulation of ER stress gene signature in TAGLN2 hi CD8 + tumor-infiltrating effector memory T cells (T EM ). NES, normalized enrichment score. p , Correlation analysis for Xbp1s versus TAGLN2 mRNA expression levels in CD8 + T cells residing in the ascites of HGSOC patients. Data were normalized to ACTB ( n = 16). q , Correlation of XBP1s versus TAGLN2 protein expression in CD45 + CD19 − CD3 + CD8 + T cells from the ascites of HGSOC patients ( n =14). Data are presented as mean ± s.e.m. a,b,d,f,h,k, One-way ANOVA with Tukey’s multiple comparisons test. e,g,j,l,m , Two-tailed unpaired Student’s t-test. p,q , Spearman’s rank correlation test, Spearman coefficient ( r ) with exact P-value (two-tailed). P <0.05 is considered statistically significant and exact P-values are shown. The ‘ n ’ values represent biologically independent samples. gMFI, Geometric mean fluorescence intensity.

Article Snippet: Expression constructs used for luciferase-based assays are XBP-1 2: pFLAG.XBP1p.CMV2 (Addgene plasmid # 21833) and Flag-p50(1–435) (Addgene plasmid # 44747) while reporter construct used is pGL3-Tagln2 promoter (−738 to +134).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Cell Culture, Binding Assay, Control, Negative Control, Real-time Polymerase Chain Reaction, Luciferase, Construct, Transfection, Two Tailed Test, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Regulation of PLPP3 gene expression by NF-κB family transcription factors

doi: 10.1074/jbc.RA119.009002

Figure Lengend Snippet: Identification and functional validation of ReREs in the human PLPP3 promoter. A, schematic of the localization of the three RelA/p50 and RelB/p52 dimer binding sites in the PLPP3 promoter. The relative locations of binding sites are identified by their distance (in base pairs) from the transcriptional start site. The core consensus NF-κB (RelA/RelB) binding sites (ReRE1–ReRE3) are indicated in blue bold (WT sequence), and changes made to generate mutant (MT1–MT3) variants of these sites are highlighted in lowercase red bold. B, normalized luciferase activity in HEK293T cells transfected with constructs containing mutated ReREs (either single site mutants or combination site mutants, as denoted in the right panel; WT sites are denoted by solid diamonds, and mutant sites are denoted by open diamonds) compared with the activity of the WT promoter reporter vector. Summarized data (mean ± S.D.) are shown from three independent transfection experiments. The p values were calculated using one-way ANOVA with Greenhouse–Geisser correction and Tukey's multiple comparisons tests. **p ≤ 0.01, ***p ≤ 0.001.

Article Snippet: Expression constructs/vectors The mammalian expression vector containing NF-κB subunits, RelB cFLAG pcDNA3, was a gift from Stephen Smale (Addgene plasmid 20017). pcDNA-FLAG-REL was a gift from Thomas Gilmore (Addgene plasmid 27253). pCMV4 p50, pCMV4p52, and pCMV4 p65 were gifts from Warner Greene (Addgene plasmids 21965, 23289, and 21966, respectively).

Techniques: Functional Assay, Biomarker Discovery, Binding Assay, Sequencing, Mutagenesis, Luciferase, Activity Assay, Transfection, Construct, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of PLPP3 gene expression by NF-κB family transcription factors

doi: 10.1074/jbc.RA119.009002

Figure Lengend Snippet: Ectopic overexpression of NF-κB component proteins and stimulation of cells with 18:1 LPA increases LPP3 protein, enzymatic activity, and mRNA levels. A–H, LPA18:1 treatment increases the levels of RelB (A) and LPP3 protein (B) in a DISP fraction and LPP3 mRNA levels (C and D) in PMA-differentiated THP-1 cells. Increases in LPP3 protein (D and E) and PLPP3 mRNA levels (F and G) in HEK293 cells were also observed after ectopic overexpression of P50/RelA or P52/RelB. Quantification of mRNA expression (D and G) depicts mean normalized ratio values ± S.D. from three independent experiments. LPA 18:1 treatment increases LPP activity in the detergent-extractable membrane protein fraction from PMA-differentiated THP-1 cells, and this increase is blocked by pretreatment of the cells with the NF-κB inhibitor parthenolide (H). The p values were calculated using an ordinary one-way ANOVA with Tukey's multiple comparisons test where necessary. ***, p ≤ 0.001. DSP, detergent-soluble particulate.

Article Snippet: Expression constructs/vectors The mammalian expression vector containing NF-κB subunits, RelB cFLAG pcDNA3, was a gift from Stephen Smale (Addgene plasmid 20017). pcDNA-FLAG-REL was a gift from Thomas Gilmore (Addgene plasmid 27253). pCMV4 p50, pCMV4p52, and pCMV4 p65 were gifts from Warner Greene (Addgene plasmids 21965, 23289, and 21966, respectively).

Techniques: Over Expression, Activity Assay, Expressing, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Regulation of PLPP3 gene expression by NF-κB family transcription factors

doi: 10.1074/jbc.RA119.009002

Figure Lengend Snippet: Schematic of the pathway by which LPA increases LPP3 expression. Our data suggest that LPA activates the PI3K–AKT pathway through LPA receptors (LPA1/3) to increase PI3K–AKT activity, which then activates NF-κB through the canonical pathway involving dissociation and ubiquitin-dependent degradation of inhibitory IκB protein, resulting in nuclear translocation of RelA/P50 or RelB/P52 transcriptional activator complexes. These activated NF-κB complexes then bind to the Rel response elements in the PLPP3 promoter that were identified in this study to initiate PLPP3 gene transcription. GPCR, G protein–coupled receptors.

Article Snippet: Expression constructs/vectors The mammalian expression vector containing NF-κB subunits, RelB cFLAG pcDNA3, was a gift from Stephen Smale (Addgene plasmid 20017). pcDNA-FLAG-REL was a gift from Thomas Gilmore (Addgene plasmid 27253). pCMV4 p50, pCMV4p52, and pCMV4 p65 were gifts from Warner Greene (Addgene plasmids 21965, 23289, and 21966, respectively).

Techniques: Expressing, Activity Assay, Ubiquitin Proteomics, Translocation Assay