Journal: Biomolecules

Article Title: The Conserved Transcriptional Activation Activity Identified in Dual-Specificity Tyrosine-(Y)-Phosphorylation-Regulated Kinase 1

doi: 10.3390/biom13020283

Figure Lengend Snippet: The on–off mechanism of transcriptional activation activity was conserved in HsDYRK1A. ( A ) The transcriptional activation ability was found in HsDYRK1A. The homologous regions of DYRK1 from diverse species with CrDYRK1 (isoform I) activation domain (residues 427–647) were amplified and used for function analysis in a yeast system, respectively. The result showed that human HsDYRK1A (residues 485–763) and another ascidian species C. savignyi CsDYRK1 (residues 393–626) presented transcriptional activation activity but HsDYRK1B (residues 437–629) did not. The homologous region (residues 629–1047) of DYRK1A in Drosophila (also called MNB) did not show the transcriptional activation activity either. ( B ) N-terminal of HsDYRK1A contained a repression domain. N-terminal (residues 1–69) could repress the transcriptional activation activity of HsDYRK1A (residues 485–763). Left panel represents the vector construction and right panel indicates the yeast assay results in ( A , B ).

Article Snippet: In the RNA-seq assay, HsDYRK1A (residues 485–763) fragment was amplified and ligated to pEGFP-N1 vector (Addgene, Watertown, MA, USA).

Techniques: Activation Assay, Activity Assay, Amplification, Plasmid Preparation

Journal: Biomolecules

Article Title: The Conserved Transcriptional Activation Activity Identified in Dual-Specificity Tyrosine-(Y)-Phosphorylation-Regulated Kinase 1

doi: 10.3390/biom13020283

Figure Lengend Snippet: The activation domain of HsDYRK1A might be involved in DYRK1A-related intellectual disability syndrome. We collected the data from the UniProt database ( https://www.uniprot.org/uniprotkb/Q13627/entry ). Some mutations were correlated to DYRK1A-related intellectual disability syndrome. The red represents the mutation-associated DYRK1A-related intellectual disability syndrome. The green represents the mutation of uncertain significance.

Article Snippet: In the RNA-seq assay, HsDYRK1A (residues 485–763) fragment was amplified and ligated to pEGFP-N1 vector (Addgene, Watertown, MA, USA).

Techniques: Activation Assay, Mutagenesis

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1 + fibroblast and CCR8 + Treg cell interactions

doi: 10.1007/s00262-022-03196-3

Figure Lengend Snippet: LLC-exo upregulate CCL1 secretion by lung fibroblasts. A Lung fibroblasts were stimulated for 24 h with either LLC-exo or PBS and inflammatory cytokines in the culture supernatant were detected using mouse cytokine microarrays. B CCL1 protein concentrations in LLC-exo stimulated fibroblasts were analyzed by mouse CCL1 ELISA. C Ccl1 mRNA levels in LLC-exo treated lung fibroblasts were detected by qRT-PCR. D CCL1 expression in LLC-exo pre-treated lungs and co-localization with α-SMA+ lung fibroblasts were determined by immunofluorescence staining; scale bar, 100 μm. Data represent the mean ± SD (n = 5). **P < 0.01 (Student’s t-test)

Article Snippet: To detect CCL1 and CCR8 expression in the lung or fibroblasts, lung sections were stained overnight at 4 °C with the following primary antibodies: CCL1 (RD system, MAB8451-SP, 1:300), CCR8 (ABclonal, A4288, 1:200) and α-SMA (Abcam, ab5694, 1:200).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1 + fibroblast and CCR8 + Treg cell interactions

doi: 10.1007/s00262-022-03196-3

Figure Lengend Snippet: LLC-exo induces Treg differentiation via the CCL1-CCR8 axis. A MPF were stimulated with either PBS or LLC-exo for 24 h and the culture media were collected (PBS and LLC-exo MPF CM, respectively). Splenic T cells were co-cultured with the LLC-exo MPF CM in the presence and absence of ZK756326 (5 μg/ml, daily) for 4 days. B, C The number of CD4+Foxp3+ Tregs was analyzed by flow cytometry; data represent the mean ± SD (n = 5). D Schematic diagram of the in vivo LLC-exo MPF CM and ZK756326 treatment experiment. E, F The proportion of Tregs in the lungs of mice in each group was determined by flow cytometry; data represent the mean ± SD (n = 5). (G) Immunofluorescence staining of CD4+Foxp3+ Tregs in LLC-exo CM ± ZK756326 treated mouse lung: CD4 (red), Foxp3 (green). Nuclei were stained with DAPI. Scale bars, 100 µm. *P < 0.05, **P < 0.01 (one-way ANOVA)

Article Snippet: To detect CCL1 and CCR8 expression in the lung or fibroblasts, lung sections were stained overnight at 4 °C with the following primary antibodies: CCL1 (RD system, MAB8451-SP, 1:300), CCR8 (ABclonal, A4288, 1:200) and α-SMA (Abcam, ab5694, 1:200).

Techniques: Cell Culture, Flow Cytometry, In Vivo, Immunofluorescence, Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Tumor-derived exosomes drive pre-metastatic niche formation in lung via modulating CCL1 + fibroblast and CCR8 + Treg cell interactions

doi: 10.1007/s00262-022-03196-3

Figure Lengend Snippet: Schematic diagram of the proposed mechanism by which TDEs drive tumor cell colonization of the lung via fibroblast CCL1 production and Treg CCR8 activation

Article Snippet: To detect CCL1 and CCR8 expression in the lung or fibroblasts, lung sections were stained overnight at 4 °C with the following primary antibodies: CCL1 (RD system, MAB8451-SP, 1:300), CCR8 (ABclonal, A4288, 1:200) and α-SMA (Abcam, ab5694, 1:200).

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Gut-derived memory γδ T17 cells exacerbate sepsis-induced acute lung injury in mice

doi: 10.1038/s41467-024-51209-9

Figure Lengend Snippet: a Heatmap showing the chemokine expression signature in sham-operated and CLP mice (n = 4). b The protein levels of CCL1 in the murine lung after combined prophylactic and therapeutic administration of S-KT or 0.9% saline (n = 6). c Transwell assay was used to assess the ability of CCL1 to chemotaxis sorted-γδ T cells from spleen mononuclear cells (n = 6). d CCR8 expression by intestinal γδ T cells in sham-operated and CLP mice was measured by FCM analysis (n = 6 for sham, 7 for CLP). Sorted-γδ T cells from spleen mononuclear cells in sham-operated or CLP mice were co-cultured with AMs. Expression of e IL-17A and f IFN-γ by γδ T cells was measured by FCM analysis (n = 3). g Heatmap of the top 29 up-regulated DEGs in the lung between the sham-operated mice and CLP mice (n = 3). h The protein levels of the components of the Wnt signaling pathway (Wnt5a, FZD5, β-catenin, and lymphoid enhancer-binding factor 1 (Lef1)) in murine lungs after combined prophylactic and therapeutic administration of S-KT or 0.9% saline (n = 6). i Average plots and heat maps showing the signals at the TSS in the ChIP-seq data sets. j Pie charts showing the distribution of all Lef1 peak locations. k Distal intergenic region-specific binding peaks between Lef1 and CCL1 gene. Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant. P values were calculated using one-way ANOVA followed by Tukey’s multiple-comparison test ( b , h ), and two-sided Student’s unpaired t-test ( c – f ). Source data and exact P values are provided as a Source Data file.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against IL-17A (1:1000, Cat# sc-374218, Santa Cruz), IL-6 (1:1000, Cat# ab229381, Abcam), IL-1β (1:1000, Cat# A1112, ABclonal), Mucin-2 (MUC-2, 1:1000, Cat# 27675-1-AP, Proteintech), ZO-1 (1:1000, Cat# 21773-1-AP, Proteintech), Occludin (1:1500, Cat# 27260-1-AP, Proteintech), Wnt5a (1:1000, Cat# 619919, Zen Bio), FZD5 (1:1000, Cat# 222996, Zen Bio), β-catenin (1:2000, Cat# 51067-2-AP, Proteintech), Lef1 (1:500, Cat# 14972-1-AP, Proteintech), TCF1/7 (1:1000, Cat# A20835, ABclonal), anti-human CCL1 (1:1000, Cat# A10670, ABclonal), anti-mouse CCL1 (1:1000, Cat# MAB845, R&D Systems), GAPDH (1:2000, Cat# 60004-1-Ig, Proteintech) and β-actin (1:2000, Cat# 66009-1-Ig, Proteintech).

Techniques: Expressing, Saline, Transwell Assay, Chemotaxis Assay, Cell Culture, Binding Assay, ChIP-sequencing, Comparison

Journal: Nature Communications

Article Title: Gut-derived memory γδ T17 cells exacerbate sepsis-induced acute lung injury in mice

doi: 10.1038/s41467-024-51209-9

Figure Lengend Snippet: a The protein expression of β-catenin, Lef1, TCF1/7 and b CCL1 in the lung (n = 3 for Lef1, 6 for TCF1/7 and CCL1). c A diagram of MH-S cell line treated with iCRT14 or vehicle followed by stimulation with LPS or PBS for 24 h in vitro to analyze the protein level of CCL1. d The protein level of CCL1 in MH-S cells (n = 7). e Gating strategy and frequency for Kaede red + γδ T17 cells in the lung of Kaede-tg septic mice treated with CCL1- shRNA (Adv-shRNA (CCL1)) or adenovirus-negative control (Adv-NC) (n = 6). f Gating strategy and frequency of Kaede red + γδ T17 cells in the lung of Kaede-tg septic mice treated with anti-CCL1 mAb or control IgG (n = 6). g Gating strategy and quantification of γδ T cells in the small intestine (n = 8). h Representative FCM plots and absolute number of Kaede red + γδ T17 cells in the lung of Kaede-tg septic mice treated with vehicle or iCRT14 (n = 6). i , j Representative H&E sections of lung and histological injury scores (n = 7). 20×, Scale bar = 100 μm. 40×, Scale bar = 50 μm. k The levels of IL-17A (n = 10 for sham + vehicle, 7 for CLP + vehicle, 6 for CLP + iCRT14), IL-6 (n = 10 for sham + vehicle, 8 for CLP + vehicle and CLP + iCRT14) and total protein (n = 6) in the BALF. l The protein level of IL-17A and IL-6 in the lung (n = 6). Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ns not significant. P values were calculated using one-way ANOVA followed by Tukey’s multiple-comparison test ( a , b , d , k , l ), and two-sided Student’s unpaired t-test ( e – h , j ). Source data and exact P values are provided as a Source Data file.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies against IL-17A (1:1000, Cat# sc-374218, Santa Cruz), IL-6 (1:1000, Cat# ab229381, Abcam), IL-1β (1:1000, Cat# A1112, ABclonal), Mucin-2 (MUC-2, 1:1000, Cat# 27675-1-AP, Proteintech), ZO-1 (1:1000, Cat# 21773-1-AP, Proteintech), Occludin (1:1500, Cat# 27260-1-AP, Proteintech), Wnt5a (1:1000, Cat# 619919, Zen Bio), FZD5 (1:1000, Cat# 222996, Zen Bio), β-catenin (1:2000, Cat# 51067-2-AP, Proteintech), Lef1 (1:500, Cat# 14972-1-AP, Proteintech), TCF1/7 (1:1000, Cat# A20835, ABclonal), anti-human CCL1 (1:1000, Cat# A10670, ABclonal), anti-mouse CCL1 (1:1000, Cat# MAB845, R&D Systems), GAPDH (1:2000, Cat# 60004-1-Ig, Proteintech) and β-actin (1:2000, Cat# 66009-1-Ig, Proteintech).

Techniques: Expressing, In Vitro, shRNA, Negative Control, Control, Comparison

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 8 Receptors of the chemokines expressed on CD3+ T cells and T cell count migrating across the transwell after blocking CCL1/2/20 in the superna tant of coculture medium. (A) The expressions of the chemokine receptors on CD3+CD56− T cells were detected using flow cytometry (n = 3 for all mol ecules). The representative images are shown. (B) Summary data of the cumulative numbers of T cells determined using a fluorescence cell analyzer after being harvested from the lower chambers, which were added into blocking antibodies against CCL1 (0.25 µg/mL), CCL2 (40 ng/mL), or CCL20 (10 µg/ mL) (n = 3 for each). Data are shown as mean ± SD. Statistical significance was determined using an unpaired t-test. *p < 0.05. Abbreviations: CXCR: C-X-C chemokine receptor; CCR:C-C chemokine receptor; XCR:X-C chemokine receptor

Article Snippet: For blocking experiments, blocking antibodies against CCL1 (0.25 μg/mL; MAB272; R&D Systems), CCL2 (40 ng/mL; MAB679; R&D Systems), and CCL20 (10 μg/ mL; MAB360; R&D Systems) were added into the conditioned media in the lower chambers.

Techniques: Cell Counting, Blocking Assay, Flow Cytometry, Fluorescence

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Il1a , Ccl5 , and Ccl1 mRNA levels in the dorsal horn of female mice. n = 5 to 8. ( B ) Immunofluorescence image of IL-1α (green) in vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). ( C ) Double immunofluorescence staining of CCL1 (green) and vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). ( D and E ) Coimmunostaining image of F4/80 (green) and CD31 (red) in the spinal dorsal horn and the statistical graph. Scale bar, 200 μm. n = 5. ( F ) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. ( G ) Immunofluorescence image shows IL-1R1 (red) expression in macrophages (green). Scale bar, 10 μm. ( H and I ) Intrathecal IL-1R1 injection inhibited the up-regulation of F4/80 (green) and CD31 (red) in the near vascular of the spinal dorsal horn. Scale bar, 200 μm (H). The number of F4/80 high cells in the dorsal horn of the spinal cord [(I), left]. The percentage of F4/80 high area among CD31 + area [(I), right]. n = 4 to 5. ( J and K ) Immunostaining shows F4/80 (green) and CD31 (red) expression in the spinal dorsal horn. Scale bar, 200 μm (J). The number of F4/80 high cells in the dorsal horn of the spinal cord [(K), left]. The percentage of F4/80 high area among CD31 + area [(K), right]. n = 5. ( L ) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. Significance: * P < 0.05, # P < 0.05, ** P < 0.01, and ## P < 0.01.

Article Snippet: For recombinant proteins IL-1α (10 μg/kg; R&D Systems, 400-ML-025/CF) and CCL1 (5 μg/kg; MCE, HY-P73913), mice were administered intraspinally (bilateral administration, the lumbar enlargement of the spinal cord).

Techniques: Immunofluorescence, Double Immunofluorescence Staining, Injection, Expressing, Immunostaining

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Coimmunofluorescence of CCL1 (green) and macrophages (purple) on day 3 following BTZ treatment in female mice. Scale bar, 50 μm. ( B ) Quantification of CCL1 fluorescence intensity in macrophages. n = 5. ( C ) Coimmunofluorescence of CCR8 (red) and NeuN (marker of neuron, green). Scale bars, 20 μm. ( D ) Coimmunofluorescence of CCR8 (red) and glial fibrillary acidic protein (GFAP; a marker of astrocyte, green). Scale bars, 20 μm. ( E ) Representative traces (left) and statistical data (right) for the action potential firing recorded in spinal cord lamina II neurons. n = 4 mice. ( F ) Intrathecal injection of CCL1-neutralizing antibody attenuated the mechanical allodynia induced by BTZ. n = 5 to 7. ( G ) CCL1 increased the action potentials of neurons in female mouse spinal slices. n = 6. ( H ) Intraspinal CCL1 injection reduced the mechanical withdrawal threshold. n = 5. ( I ) R243 preincubation blocked the CCL1-induced action potential increase in female mouse spinal slices. n = 6. ( J ) Intrathecal injection of R243 attenuated the mechanical allodynia induced by BTZ. n = 5. ( K ) Intrathecal injection of R243 attenuated the mechanical allodynia induced by recombinant CCL1. n = 5. ( L ) Recombinant CCL1 increased the action potentials in astrocyte CCR8 knockdown mouse spinal slices. n = 5. ( M ) Intraspinal CCL1 injection reduced the mechanical withdrawal threshold in astrocyte CCR8 knockdown female mice. n = 5. Significance: not significant (n.s.), * P < 0.05 and ** P < 0.01.

Article Snippet: For recombinant proteins IL-1α (10 μg/kg; R&D Systems, 400-ML-025/CF) and CCL1 (5 μg/kg; MCE, HY-P73913), mice were administered intraspinally (bilateral administration, the lumbar enlargement of the spinal cord).

Techniques: Fluorescence, Marker, Injection, Recombinant, Knockdown

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Immunofluorescence imaging showed BTZ-activated astrocytes (turquoise) in close proximity to macrophages (green) and vascular endothelial cells (red). Scale bars, 20 μm. ( B ) Schematic of two-photon imaging configuration. ( C ) Representative image showed the increased GCaMP6s (green) in spinal cord astrocytes (SR101, red) following CCL1 incubation. Scale bar, 150 μm. ( D ) Example signal trace (left) and histogram of area under the curve (AUC) (right) of calcium signaling value from the spinal dorsal horn astrocyte in mice treated with CCL1. n = 3. ( E to G ) R243 incubation prevented CCL1-induced calcium signaling increase in GCaMP6s + astrocytes. Representative image (E) and signal trace (F) show R243 blocked CCL1-induced calcium up-regulation. Scale bar, 20 μm. AUC quantification (G). n = 3. ( H to J ) Response of astrocytes to recombinant CCL1 during bath application of TTX (1 μM). Representative image (H) and signal trace (I) showed that TTX did not inhibit the increase in intracellular calcium signaling induced by CCL1. Scale bar, 20 μm. AUC quantification (J). n = 3. ( K to M ) Application of CNO increased the astrocyte intracellular calcium signaling in the female with injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. Scale bar, 20 μm. ( N ) The action potential number in female mouse spinal cord slice was increased by CNO application. Representative trace (left) and statistical data (right) were shown. n = 3. ( O ) Application with CNO decreased the paw withdrawal threshold in female mice with intraspinal injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. n = 5. Significance: not significant (n.s.) and * P < 0.05.

Article Snippet: For recombinant proteins IL-1α (10 μg/kg; R&D Systems, 400-ML-025/CF) and CCL1 (5 μg/kg; MCE, HY-P73913), mice were administered intraspinally (bilateral administration, the lumbar enlargement of the spinal cord).

Techniques: Immunofluorescence, Imaging, Incubation, Recombinant, Injection

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Increased ICAM-1 promotes monocyte adhesion to vascular endothelial cells following BTZ treatment. ( B ) IL-1α/ICAM-1 collaboratively promote monocyte transmigration across the vascular wall. ( C ) Infiltrated macrophage-released CCL1 enhances neuron excitability directly or indirectly via the activated astrocytes.

Article Snippet: For recombinant proteins IL-1α (10 μg/kg; R&D Systems, 400-ML-025/CF) and CCL1 (5 μg/kg; MCE, HY-P73913), mice were administered intraspinally (bilateral administration, the lumbar enlargement of the spinal cord).

Techniques: Transmigration Assay