proteome profilertm array mouse xl cytokine array kit  (R&D Systems)

Journal: Nature Communications

Article Title: Loss of tumor suppressors promotes inflammatory tumor microenvironment and enhances LAG3+T cell mediated immune suppression

doi: 10.1038/s41467-024-50262-8

Figure Lengend Snippet: A schematic analysis for ATAC and RNA-seq datasets. B RNA-seq Venn diagram showing overlapped genes from in vitro cultured 4T1 cells with sgRNA mediated deletion of Nf1, Tsc1, or Tgfbr2 compared with control 4T1-C1 cells. Cut-off: FC > 1.5, P < 0.05. C ATAC-seq Venn diagram (left) and heatmap (right) showing common differential peaks for all three TS KO vs the control cells. Cut-off: Differential ATAC peaks p < 0.01, FC < −2 and >2. D Increased ATAC peaks for IL6, JAK3 and decreased ATAC peaks for CCL2 that are common for all three TS KO cells, Gapdh as control. E Relative expression ( RT-PCR) of IL6, JAK3, IDO1, and CCL2 comparing sgNf1, sgTsc1 or sgTgfbr2 vs control ( n = 4). (F) IL6 ( n = 3) and IDO1 ( n = 3) ELISA (left two panels), JAK3 Western, and CCL2 Cytokine Array and quantitative data (right two panels, IL-23 as control) from sgNf1, sgTsc1 or sgTgfbr2 compared with control. G Western blots of pSTAT3, pSTAT6, pJAK3 and JAK3 comparing sgNf1, sgTsc1 or sgTgfbr2 4T1 cells with C1 control. H RT-PCR of IL6 or IDO1 of cells treated with a pan JAK or a JAK3 specific inhibitor or IL6-neuAb ( n = 3). All graphs show mean ± s.d. Average of each biological replicate was plotted, and statistical significance was determined by two-tailed Student’s t -test for ( E ), ( F ), and ( H ). * p < 0.05; ** p < 0.01. Exac t p -values for ( E ), ( F ), and ( H ) are provided in a source data file.

Article Snippet: For protein concentration detection, supernatant and/or cell lysate protein extractions were examined using the Mouse IDO1(Indoleamine 2,3-dioxygenase 1) ELISA Kit (FineTest, EM0653), QuantikineTM ELISA Mouse IL6 Immunoassay (R&D Systems, M6000B), TGF-β1 Quantikine ELISA (R&D Systems, DB100B,), Proteome ProfilerTM Array Mouse XL Cytokine Array Kit (Cat. ARY028, R&D Systems) according to manufacturer instructions.

Techniques: RNA Sequencing Assay, In Vitro, Cell Culture, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Neutralizing Assay, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Endothelial PDGF-D contributes to neurovascular protection after ischemic stroke by rescuing pericyte functions

doi: 10.1007/s00018-024-05244-w

Figure Lengend Snippet: Brain pericyte survival and vascular protective properties are stimulated by PDGF-D. ( a ) Schematic illustration of the experimental design of primary human brain vascular pericytes (HBVP) exposition to oxygen and glucose deprivation (OGD) followed by rh-PDGF-D stimulation. ( b ) Western blot analysis showing BCL2 expression in vehicle (VEH)-, rh-PDGF-D 40 ng/mL (P40)- or rh-PDGF-D 80 ng/mL (P80)-treated HBVP, 24 h after OGD. ( c ) Western blot analysis showing NOTCH3 expression in VEH, P40 or P80 groups, 24 h after OGD. ( d ) Representative brightfield images of VEH and P80 groups, immediately upon or 24 h after wound formation (white dashed lines) under normoxic (NOR) or OGD conditions. ( e ) Analysis of wound closure of VEH and P80 groups under NOR or OGD conditions at 24 h, represented as percent of control (baseline, 0 h). ( f ) Representative images of the proteome Profiler human angiogenesis array membranes analyzing the expression of factors implicated in vascular remodeling in VEH and P80 groups exposed to NOR or OGD, 24 h after stimulation. Blue rectangles highlight downregulated factors and red rectangles highlight upregulated factors. ( g ) Heatmap illustration showing differentially regulated factors in OGD-exposed HBVP upon PDGF-D stimulation. ( h ) Analysis of VEGF, uPA and IGFBP1 expression in NOR- and OGD-exposed HBVP upon stimulation of PDGF-D. ( i ) Representative brightfield images of tubes formed by immortalized human brain endothelial cells (iHBEC) without treatment (EC), treated with P80 (EC + P80) or with CM of OGD-exposed HBVP (PC) stimulated with P80 (EC + CM), or co-cultured with HBVP (PC) without treatment (EC/PC), treated with P80 (EC/PC + P80) or with CM of OGD-exposed PC stimulated with P80 (EC/PC + CM), 8 h after plating cells on ECM. (j) Analysis of the density of tubes formed under the different conditions. ( b-e, j ) Data are boxplot with min/max ( n = 3–4/groups). * P < 0.05/ ** P < 0.01/ *** P < 0.001/ **** P < 0.0001 compared to other different condition ( (b) Kruskal-Wallis/Dunn’s test, ( c, e, j ) one-way ANOVA/Tukey’s test or ( h ) unpaired two tailed t-test). H, hour

Article Snippet: Proteome ProfilerTM human angiogenesis array kit (R&D Systems; ARY007) was used to simultaneously profile the expression of 55 angiogenesis-related proteins of unstimulated and PDGF-D-stimulated HBVP under normoxic or OGD conditions at 24 h, following manufacturer’s instructions.

Techniques: Western Blot, Expressing, Control, Cell Culture, Two Tailed Test

Journal: Redox Biology

Article Title: Nuclear translocation of metabolic enzyme PKM2 participates in high glucose-promoted HCC metastasis by strengthening immunosuppressive environment

doi: 10.1016/j.redox.2024.103103

Figure Lengend Snippet: Identification of differentially expressed chemokines under high glucose conditions and their relationship with immunosuppressive microenvironment in HCC tissues (A) The comparative analysis of differentially secreted chemokines between HG-CM and NG-CM using a human cytokine/chemokine microarray (upper panel). The semi-quantitative analysis of the differential secretion of chemokines between HG-CM and NG-CM was conducted using fold change analysis (bottom panel). (B) Flow cytometry analysis of CD4 + CD25 + FoxP3 + Treg cells, CD68 + CD163 + Macrophage M2 cells, and CD8 + T cells in HCC tissues from four distinct groups (HCC-DM, HCC-Control, HCC-DM + INS, and HC-DM + CANA). The average prevalence of CD4 + CD25 + FoxP3 + Treg cells, CD68 + CD163 + Macrophage M2 cells, and CD8 + T cells was calculated. Data are shown as means ± SD, n = 3∼4 per group (bottom panel). (C) The analysis of the association between the leading differentially expressed chemokine (RARRES2) and infiltrated immunosuppressive cells (Macrophage M2 and Treg cells) using TCGA-HCC data (D) Representative IHC staining images displayed the expression of chemerin in HCC tissues from four animal groups (left panel). The average density of chemerin in HCC tissues was quantified by analyzing 6 images per group using ImageJ software (right panel). (E) Western blot analysis showed the expression of chemerin and CXCL16 in HCC cells cultured in media with normal glucose, moderate glucose, and high glucose concentrations (left panel). The protein expression levels were quantified (right panel). (F) Survival analysis of RARRES2 expression using TCGA-HCC data. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: HG-CM, high glucose-conditioned media; NG-CM, normal glucose-conditioned media; MG, moderate glucose.

Article Snippet: The conditioned media from HCC cells cultured in high glucose and normal glucose conditions (HG-CM and NG-CM) for 48 h were collected respectively, and their differentially expressed chemokines were analyzed using a Proteome ProfilerTM Human Chemokine Array Kit (R&D Systems, Cat. No. ARY017) in accordance with the instructions and the published studies [ , , ].

Techniques: Microarray, Flow Cytometry, Control, Immunohistochemistry, Expressing, Software, Western Blot, Cell Culture