Journal: Scientific reports

Article Title: Ischemic stroke outcome after promoting CD4+CD25+ Treg cell migration through CCR4 overexpression in a tMCAO animal model.

doi: 10.1038/s41598-024-60358-2

Figure Lengend Snippet: Figure 2. Confirmation of the selected receptors in cytotoxic T cells and Treg cells through immunocytochemistry (ICC) and western blot. (a) ICC confocal imaging results showing the expression of the screened receptors: CXCR4, CD4, CCR4, and CCR10 on cytotoxic T cells and Treg cells, as well as CD8α or FoxP3, respectively. (b) The corrected total cell fluorescence (CTCF) was measured with ImageJ and normalized against either CD8α or FoxP3, respectively. (c) Western blot results of the receptors listed above, as well as β-actin as a reference in cytotoxic T cells and Treg cells. Each receptor from each cell type was ran in different gels and transferred onto separate membrane (full length gels can be viewed in the Supplementary Fig. S2). (d) The above western blot band intensity for each receptor was measured using ImageJ and normalized against the β-actin bands in cytotoxic T cells and Treg cells. Asterisks denote the significance of data from p-values obtained by performing the Two-way ANOVA and Sidak’s multiple comparison (*p ≤ 0.05, **p ≤ 0.01, ****p < 0.0001, all n = 3).

Article Snippet: The CCR4 inhibitor (sc-221406) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas), and the CXCR4 inhibitor (WZ811) was obtained 3 Vol.

Techniques: Immunocytochemistry, Western Blot, Imaging, Expressing, Fluorescence, Membrane, Comparison

Journal: Scientific reports

Article Title: Ischemic stroke outcome after promoting CD4+CD25+ Treg cell migration through CCR4 overexpression in a tMCAO animal model.

doi: 10.1038/s41598-024-60358-2

Figure Lengend Snippet: Figure 3. The Treg cell MSCV-CCR4 retrovirus transduction showed an increase in CCR4 expression, further increasing the migration into the OGD media. (a) The MSCV retrovirus vector construct was designed via VectorBuilder. The mCcr4 sequence was linked with the EGFP reporter gene with a P2A linker, where the expression was controlled by the MSCV ψ+ promoter. (b) Transduction was performed following this schedule. (c) ICC imaging of the UT and TD Treg cells. (d) CTCF was measured with the ImageJ program and normalized against the FoxP3 fluorescence measurements. (e) The detailed quantification of changes in CCR4 expression was observed using FACS. (f, g) Graph of the FoxP3+ Treg cell population expressing EGFP and CCR4 highly. (h) Confocal images of Treg cells that migrated into the chamber containing OGD media. (i) The number of Treg cells that migrated into the OGD media after 48 h. Asterisks denote the significance of the p-values obtained by performing the unpaired t-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, all n = 3–5).

Article Snippet: The CCR4 inhibitor (sc-221406) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas), and the CXCR4 inhibitor (WZ811) was obtained 3 Vol.

Techniques: Transduction, Expressing, Migration, Plasmid Preparation, Construct, Sequencing, Imaging, Fluorescence

Journal: Neuro-oncology Advances

Article Title: Immune deconvolution and temporal mapping identifies stromal targets and developmental intervals for abrogating murine low-grade optic glioma formation

doi: 10.1093/noajnl/vdab194

Figure Lengend Snippet: CD8 + T cell predominance is governed by NF1/RAS regulation of Ccr4 expression. A, CD8 + T cells content in Nf1 -OPG optic nerves ( n = 10) is similar to controls ( n = 8) at 3, 4.5, 6, and 9 WOA, but increases by 12 WOA ( P < 0.0001). Nf1 +/– mice ( n = 6) have similar numbers of CD8 + T cells as controls from 3 to 12 WOA. B, CD4 + T cell content does not change in Nf1 -OPG optic nerves relative to controls at 3, 4.5, 6, 9, and 12 WOA. C, Ccl2 did not increase CD4 + T cell migration in WT or Nf1 +/– mice. Ccl12 or medium (control) does not increase CD4 + T cell migration. Ccl2 increased Nf1 +/– CD8 + T cell migration relative to WT controls ( P < 0.0001). Nf1 +/- and WT CD8 + T cells treated with medium (Control) or Ccl12 exhibit similar migration. D, Nf1 +/- and WT CD4 + T cells had similar levels of Ccr4 expression. Nf1 +/– CD8 + T cells have increased Ccr4 expression relative to their WT counterparts ( P = 0.0474). E, Ccr4 inhibition (AZD2098; 15µM) in CD8 + T cells decreases Ccl2-induced, but not Ccl12-induced, migration ( P = 0.0063). F, Nf1 +/– and WT CD4 + T cells had similar levels of Ras activity. Nf1 +/– CD8 + T cells have increased Ras activity relative to their WT counterparts ( P = 0.0363) G, Inhibition of Ras activity (10µM IN-1) reduces Ccl2-induced,but not Ccl12-induced, CD8 + T cell migration ( P = 0.0104). H, RAS activity inhibition (10µM IN-1) decreases Ccr4 expression in Nf1+/- CD8 + T cells ( P = 0.0069). I, Proposed model of Ccl2 signaling through Ccr4 in Nf1+/– CD8 + T cells.

Article Snippet: After 24h, Nf1 +/- CD8 + T cells were treated for 24 h with 10 μM Pan-RAS-Inhibitor-IN-1 (IN-1[HY-101295, MedChem Express]) or 10 μM Ccr4 inhibitor (AZD2098, Selleckchem).

Techniques: Expressing, Migration, Control, Inhibition, Activity Assay

Journal: Scientific Reports

Article Title: Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1

doi: 10.1038/srep28687

Figure Lengend Snippet: ( a ) HASMCs that were transfected with WT, S405A, S418A or S405/418A mutant cortactin expression vector and treated with vehicle or MCP1 for 8 hrs were analyzed by immunoblotting for p21Cip1 levels and the blot was reprobed for GFP or β-tubulin to show the overexpression of WT or mutant cortactin or normalization. ( b ) All the conditions were the same as in panel a except that cells were subjected to MCP1-induced DNA synthesis. ( c ) Equal amounts of protein from control and the indicated treatments were immunoprecipitated with anti-acetyl lysine or anti-phosphotyrosine antibodies and the immunocomplexes were analyzed by immunoblotting for cortactin. The same cell extracts were also analyzed by immunoblotting for cortactin. ( d ) HASMCs were treated with vehicle or MCP1 in the presence and absence of HDAC6i or HDAC8i (10 μM) for 6 hrs and equal amounts of protein from control and each treatment were immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by immunoblotting with anti-acetyl lysine antibody and normalized to cortactin. ( e ) HASMCs were treated with vehicle or MCP1 in the presence and absence of HDAC6 inhibitor for 8 hrs and equal amounts of protein from control and each treatment were analyzed by immunoblotting for p21Cip1 and normalized to β-tubulin. ( f ) The cloning strategy for cortactin expression vectors is presented and the mutant amino acid is shown in red. ( g ) HASMCs were transfected with WT or Y5F mutant cortactin expression vector, treated with vehicle or MCP1 for 8 hrs and equal amounts of protein from control and each treatment were immunoprecipitated with anti-pTyr antibody and the immunocomplexes were analyzed by immunoblotting for GFP. Same cell extracts were also analyzed by immunoblotting for GFP-CTTN and p21Cip1 and normalized to β-tubulin. ( h ) All the conditions were the same as in panel g except that cells were subjected to MCP1-induced DNA synthesis. The bar graphs in ( a – e , g , h ) represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control or WT-CTTN; **p < 0.05 vs MCP1 or WT-CTTN + MCP1. HDAC6i, HDAC6 inhibitor; HDAC8i, HDAC8 inhibitor.

Article Snippet: CCR2 antagonist (SC-202525), CCR4 antagonist (SC-221406) HDAC6 inhibitor (SC-223877), HDAC8 inhibitor PCI-34051 (SC-364566), anti-cortactin (SC-11408), anti-Gαq (SC-393), anti-Gα11 (SC-394; SC-390382), anti-GFP (SC-9996), anti-Fyn (SC-365913), anti-Lyn (SC-15), anti-p21Cip1 (SC-6246), anti-p27Kip1 (SC-528), anti-p57Kip2 (SC-1040), anti-pYes (SC-130182), anti-Yes (SC-8403) and anti-β-Tubulin (SC-9104) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Over Expression, DNA Synthesis, Control, Immunoprecipitation, Cloning

Journal: Scientific Reports

Article Title: Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1

doi: 10.1038/srep28687

Figure Lengend Snippet: ( a ) Growth-arrested HASMCs were treated with vehicle or MCP1 in the presence and absence of CCR2 antagonist (CCR2A) or CCR4 antagonist (CCR4A) for 6 hrs and the cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for pFyn levels using anti-pFyn antibodies and normalized to Fyn. Equal amounts of protein from the same cell extracts were also immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by Western blotting with anti-pTyr antibodies and normalized to cortactin. ( b ) All the conditions were the same as in panel a except that equal amounts of protein from control and each treatment were analyzed by Western blotting for p21Cip1 levels using its specific antibodies and normalized to β-tubulin. ( c ) All the conditions were the same as in panel a except that cells were treated with vehicle or MCP1 for 6 hrs and co-immunostained for p21Cip1 and 20Sα/β using their specific antibodies. ( d ) All the conditions were the same as in panel a except that cells were subjected to MCP1-induced DNA synthesis. ( e ) Growth-arrested cells were treated with vehicle or MCP1 in the presence and absence of BAPTA-AM (10 μM) for 6 hrs and cell extracts were prepared. An equal amount of protein from control and each treatment was immunoprecipitated with anti-cortactin antibodies and the immunocomplexes were analyzed by immunoblotting with anti-pTyr antibodies and normalized for cortactin. Equal amounts of protein from the same cell extracts were also analyzed by Western blotting for p21Cip1 levels and normalized to β-tubulin. The bar graphs in panels a,b,d and e represent Mean ± SD values of three independent experiments. *p < 0.05 vs vehicle control; **p < 0.05 vs MCP1.

Article Snippet: CCR2 antagonist (SC-202525), CCR4 antagonist (SC-221406) HDAC6 inhibitor (SC-223877), HDAC8 inhibitor PCI-34051 (SC-364566), anti-cortactin (SC-11408), anti-Gαq (SC-393), anti-Gα11 (SC-394; SC-390382), anti-GFP (SC-9996), anti-Fyn (SC-365913), anti-Lyn (SC-15), anti-p21Cip1 (SC-6246), anti-p27Kip1 (SC-528), anti-p57Kip2 (SC-1040), anti-pYes (SC-130182), anti-Yes (SC-8403) and anti-β-Tubulin (SC-9104) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Control, Western Blot, Immunoprecipitation, DNA Synthesis

Journal: Molecular cell

Article Title: Argonaute-miRNA Complexes Silence Target mRNAs in the Nucleus of Mammalian Stem Cells

doi: 10.1016/j.molcel.2018.07.020

Figure Lengend Snippet: ShRNA against CNOT1 and CNOT7

Article Snippet: Rabbit polyclonal CNOT7 , Proteintech , RRID:AB_2245087; Cat# 14102–1-AP.

Techniques: shRNA

Journal: Molecular cell

Article Title: Argonaute-miRNA Complexes Silence Target mRNAs in the Nucleus of Mammalian Stem Cells

doi: 10.1016/j.molcel.2018.07.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal CNOT7 , Proteintech , RRID:AB_2245087; Cat# 14102–1-AP.

Techniques: Magnetic Beads, Virus, Recombinant, Reverse Transcription, Protease Inhibitor, Plasmid Preparation, SYBR Green Assay, shRNA, Mutagenesis, Software