Journal: Hypertension

Article Title: Activation of Human T Cells in Hypertension

doi: 10.1161/hypertensionaha.116.07237

Figure Lengend Snippet: Figure 4. Effect of hypertension on myeloid differentiation in the kidney, aorta, and lymph nodes. Summary data from mice after sham or angiotensin II (Ang II) infusion (490 ng/kg per min) of non-CD3+ CD45+ cells in the kidney (A), descending, thoracic aorta (B), and thoracic lymph nodes (C). The flow cytometry gating strategy used to identify myeloid cells is shown (D). The kidney, aorta, and lymph nodes of the humanized mice all showed differentiation of the monocytes to dendritic cells (E). Myeloid differentiation was not significantly shifted in any of the tissues when we compared monocyte (CD14+CD83−) vs activated monocyte (CD14+CD83+) vs dendritic cell (CD14−CD83+) populations. 7-AAD indicates 7-aminoactinomycin D; FSC-A, forward scatter area; FSH, forward scatter height; and SSC-A, side scatter area.

Article Snippet: Antibodies used for flow cytometry in the humanized mice studies were as follows: peridinin chlorophyll protein–cyanin-5.5–conjugated (PerCP-Cy5.5) anti-CD45 mouse antibody (BDbiosciences, clone 30-F11), allophycocyanin–Hilite7–conjugated (APC-H7) anti-CD45 human antibody (BD Biosciences, clone 2D1), Phycoerythrin (PE) anti-CD3 human (BD Biosciences, clone UCHT1), Brilliant Violet 510–conjugated (BV510) anti-CD4 human antibody (BD Biosciences, clone SK3), Brilliant Violet 450–conjugated (BV450) anti-CD8 human antibody (BD Biosciences, clone RPA-T8), Fluorescein isothiocyanate (FITC) anti-CD45RO human antibody (BD Biosciences, clone UCHL1), Allophycocyanin (APC) anti-CD69 human (BD Biosciences, clone FN50), Phycoerythrin (PE) anti-CD45 human (eBiosciences, clone 2D1), Fluorescein isothiocyanate (FITC) anti-CD14 human antibody (miltenyibiotec, clone TÜK4), Brilliant Violet 510–conjugated (BV510) anti-CD83 human antibody (BD Biosciences, clone HB15e), FITC-conjugated isoketal adduct antibody (D11), APC anti-foxP3 human antibody (BD Biosciences), FITC anti-IFN-γ human antibody (BD Biosciences, catalogue #: 502505), and BV510 anti-IL-17A human antibody (BD Biosciences, Clone N49-653).

Techniques: Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Interaction of NF-κB and FOSL1 drives glioma stemness

doi: 10.1007/s00018-024-05293-1

Figure Lengend Snippet: GSEA enrichment of pathways associated with the regulation of NF-κB signaling. ( A ) The GSEA enrichment of positive regulation of NF-κB transcription factor activity. ( B ) The GSEA enrichment of IκB phosphorylation. ( C, D ) Heat maps showing the top 15 genes and last 15 genes corresponding to the positive regulation of NF-κB signaling pathways in panel A ( C ) and all 15 genes involved in κB phosphorylation signaling pathways in panel B ( D ). ( E-I ) qPCR Validation of the top 3 differentially expressed genes in panel C ( E : CD30; F : PRKCQ; and G : ADAM8) and top 2 in panel D ( H : TLR7; I : ERC1) in A172, U87MG, and PDX-L14 cells with overexpressed FOSL1. The endogenous FOSL1, TRPM7 and NF-κB protein expression levels were examined in glioma cell lines by Western blot ( J upper panel) and the correlation between FOSL1 and TRPM7 ( J middle panel), as well as between FOSL1 and NF-κB ( J lower panel) was analyzed by Pearson’s correlation in GraphPad

Article Snippet: Quantitative PCR was performed using Bio-Rad SYBR QPCR Master Mix (Bio-Rad, cat. no. 1,708,882) with the CFX Connect Real-Time PCR Detection System (Bio-Rad).

Techniques: Activity Assay, Phospho-proteomics, Protein-Protein interactions, Biomarker Discovery, Expressing, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Interaction of NF-κB and FOSL1 drives glioma stemness

doi: 10.1007/s00018-024-05293-1

Figure Lengend Snippet: The positive role of NF-κB in transactivating the FOSL1 gene and amplifying its expression. ( A ) A172, U87MG, and PDX-L14 cells transfected with pCMV-p65 were assessed for total and phosphorylated NF-κB and FOSLl1 protein levels via Western blot. ( B ) Similar cells treated as in ( A ) underwent an ELISA to measure relative NF-κB activity in nuclear fraction lysates (* indicates p < 0.05 compared to the control using a Student’s t-test). ( C ) The pCMV p65 were transiently transfected into A172, U87MG, and PDX-L14 cells alongside wt-FOSL1 luc or mutant M1-5 constructs. Subsequently, luciferase activity was analyzed after a 24-hour incubation. ( D-F ) ChIP-qPCR analysis of direct binding to the FOSL1 promoter in A172 cells ( D ), U87MG cells ( E ), and PDX-L14 cells ( F ) transfected with pCMV-p65 and vector. ChIP-qPCR results were analyzed by evaluating the signal of enrichment of over noise normalized to input. DNA levels were normalized to relative inputs ( n = 3 independent experiments; * indicates p < 0.05 among groups using one-way ANOVA). ( G ) Western blot analysis of NF-κB and FOSL1 protein levels in A172, U87MG, and PDX-L14 cells transfected with siNF-κB p65. ( H ) A similar treatment in ( G ), followed by an ELISA for relative NF-κB activity in nuclear fraction lysates (* indicates p < 0.05 compared to control using a Student’s t-test). ( I ) Transient transfection of siNF-κB p65 into A172, U87MG, and PDX-L14 cells alongside wt-FOSL1 luc or mutant M1-5 constructs, followed by analysis of luciferase activity as described in ( C )

Article Snippet: Quantitative PCR was performed using Bio-Rad SYBR QPCR Master Mix (Bio-Rad, cat. no. 1,708,882) with the CFX Connect Real-Time PCR Detection System (Bio-Rad).

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Mutagenesis, Construct, Luciferase, Incubation, ChIP-qPCR, Binding Assay, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Interaction of NF-κB and FOSL1 drives glioma stemness

doi: 10.1007/s00018-024-05293-1

Figure Lengend Snippet: Overexpression of FOSL1 in GBM positively influences NF-κB. ( A-D ) A172, U87MG, and PDX-L14 cells were transfected with GFP-tagged human FOSL1 (FOSL1) and controls (Ctrl) followed by assaying protein expression of FOSL1 and GFP by Western blot ( A ), mRNA expression of FOS1 by qPCR ( B ), GFP positive cell by flow cytometry ( C ), and by immunofluorescence staining ( D : A172-left, U87MG-middle, and PDX-L14-right). ( E ) The identical cellular samples, treated as detailed in ( A-D ), were subjected to ELISA analysis to quantify the relative NF-κB activity in nuclear fraction lysates (* denotes significance at p < 0.05 compared to control using a Student’s t-test). ( F ) Subsequent to the procedures outlined in ( E ), qPCR was conducted to evaluate the mRNA levels of NF-κB p65 and p50. ( G ) Following the protocols described in ( E ), Western blotting was employed to examine the levels of FOSL1 protein, along with phosphorylated (p-IKKβ) and total IKKβ, phosphorylated (p-IKBα) and total IKBα, and phosphorylated (p-P65) and total NF-κB p65. Densitometry analysis for Western blots was performed using the Image Quant program, with the corresponding results positioned below or adjacent to each image. The bar graphs represent the mean ± S.D. of three independent experiments

Article Snippet: Quantitative PCR was performed using Bio-Rad SYBR QPCR Master Mix (Bio-Rad, cat. no. 1,708,882) with the CFX Connect Real-Time PCR Detection System (Bio-Rad).

Techniques: Over Expression, Transfection, Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Control