Journal: PLoS ONE

Article Title: P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

doi: 10.1371/journal.pone.0153312

Figure Lengend Snippet: Contractions of isolated human prostate strips were induced as indicated, after addition of 10 μM FRAX48, 100 μM IPA3, or DMSO (control). Tensions have been expressed as % of contraction by highmolar KCl, being assessed before application of inhibitors or solvent. This normalization allows comparisons despite different conditions of tissues or patients, e. g. due to varying degree of BPH, different content of smooth muscle, or any other heterogeneity (compare ). Shown are means ± SEM from experiments with tissues from n = 5 (endothelin-1, EFS, each with FRAX486), n = 4 (endothelin-2), or n = 3 (EFS with IPA3) patients for each group.

Article Snippet: FRAX486 and IPA3 were obtained from Tocris (Bristol, UK), phenylephrine and norepinephrine were obtained from Sigma (Munich, Germany).

Techniques: Isolation, Control, Solvent

Journal: PLoS ONE

Article Title: P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

doi: 10.1371/journal.pone.0153312

Figure Lengend Snippet: Actin filaments were visualized by staining with FITC-coupled phalloidin, after incubation of WPMY-1 cells with DMSO, FRAX486 (1–10 μM), or IPA3 (1–10 μM) for 24 h. Shown are representative images from series with 5 independent experiments, with similar results.

Article Snippet: FRAX486 and IPA3 were obtained from Tocris (Bristol, UK), phenylephrine and norepinephrine were obtained from Sigma (Munich, Germany).

Techniques: Staining, Incubation

Journal: PLoS ONE

Article Title: P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

doi: 10.1371/journal.pone.0153312

Figure Lengend Snippet: Survival of WPMY-1 cells was assessed using CCK-8 assay, after incubation with DMSO, FRAX486 (1–10 μM), or IPA3 (1–10 μM) for 24–72 h. Shown are means ±SEM from series with 5 independent experiments for each setting (# p<0.05 vs. control).

Article Snippet: FRAX486 and IPA3 were obtained from Tocris (Bristol, UK), phenylephrine and norepinephrine were obtained from Sigma (Munich, Germany).

Techniques: CCK-8 Assay, Incubation, Control

Journal: PLoS ONE

Article Title: P21-Activated Kinase Inhibitors FRAX486 and IPA3: Inhibition of Prostate Stromal Cell Growth and Effects on Smooth Muscle Contraction in the Human Prostate

doi: 10.1371/journal.pone.0153312

Figure Lengend Snippet: Proliferation rate was assessed by EdU assay after incubation with DMSO (control), FRAX486 (1–10 μM), or IPA3 (1–10 μM) for 24 h. The number of cells showing proliferation (= red nuclei) was referred to the total number of cells (= red + blue nuclei), to correct for reduced number of cells after longer incubation periods (compare with ). Shown are representative images and means ±SEM, from series with 8 independent samples for each setting (# p<0.05 vs. control).

Article Snippet: FRAX486 and IPA3 were obtained from Tocris (Bristol, UK), phenylephrine and norepinephrine were obtained from Sigma (Munich, Germany).

Techniques: EdU Assay, Incubation, Control

Journal: mSystems

Article Title: Harmane induces apoptosis through RRM2B and suppresses colorectal cancer progression

doi: 10.1128/msystems.01704-25

Figure Lengend Snippet: Dynamic remodeling of the gut metabolome across healthy populations, patients with advanced adenoma, and CRC patients. ( A ) Heatmap showing the KEGG pathway enrichment of gut metabolites that gradually decreased from healthy individuals to advanced adenoma patients and further to CRC patients. ( B ) Three metabolites showing consistent downward concentration trends along the disease continuum. ( C ) Eight metabolites demonstrating gradually increasing concentrations from healthy states to CRC states. ( D ) Heatmap illustrating the KEGG pathway enrichment of metabolites with increasing abundances. ( E ) Chemical structure of harmane. ( F ) Cytotoxic effect of harmane on CRC cells evaluated by CCK-8 assay.

Article Snippet: Following overnight incubation, CRC cells were treated with either 100 μM harmane (MedChemExpress, USA) or a vehicle control for 24 h. After treatment, 10 μL of CCK-8 solution (Vazyme, China) was added directly to each well, and the plates were incubated at 37°C for 4 h. Cell viability was determined at OD at 450 nm using a microplate reader (Thermo Scientific, USA).

Techniques: Concentration Assay, CCK-8 Assay

Journal: mSystems

Article Title: Harmane induces apoptosis through RRM2B and suppresses colorectal cancer progression

doi: 10.1128/msystems.01704-25

Figure Lengend Snippet: Harmane suppresses proliferation, migration, and invasion and induces apoptosis in CRC cells. ( A ) Viability of HCT8 and SW480 cells treated with 100 μM harmane for 24 h determined by CCK-8 assay. ( B, C ) Proliferative capacity of CRC cells evaluated by ( B ) colony formation assay and ( C ) EdU incorporation assay after 24 h harmane treatment. Scale bar: 100 μm. ( D, E ) Migratory and invasive abilities of CRC cells assessed by ( D ) transwell migration assay and ( E ) Matrigel invasion assay following 24 h harmane treatment. Scale bar: 100 μm. ( F ) Cell cycle distribution analyzed by flow cytometry using PI staining after 24 h harmane treatment. ( G ) Induction of apoptosis measured by annexin V/PI staining and flow cytometry. All data were derived from one representative experiment out of three independent biological replicates. Results are presented as means ± SEM. Statistical significance was determined using two-way ANOVA with Sidak’s multiple comparisons test. *** P < 0.005, **** P < 0.001.

Article Snippet: Following overnight incubation, CRC cells were treated with either 100 μM harmane (MedChemExpress, USA) or a vehicle control for 24 h. After treatment, 10 μL of CCK-8 solution (Vazyme, China) was added directly to each well, and the plates were incubated at 37°C for 4 h. Cell viability was determined at OD at 450 nm using a microplate reader (Thermo Scientific, USA).

Techniques: Migration, CCK-8 Assay, Colony Assay, Transwell Migration Assay, Invasion Assay, Flow Cytometry, Staining, Derivative Assay

Journal: mSystems

Article Title: Harmane induces apoptosis through RRM2B and suppresses colorectal cancer progression

doi: 10.1128/msystems.01704-25

Figure Lengend Snippet: Harmane induces apoptosis and cell cycle arrest in CRC cells through RRM2B. ( A ) Circos plot displaying differential expression patterns of apoptosis-related genes between control and harmane-treated HCT8 cells. ( B ) Western blot analysis of apoptosis-related protein expression in CRC cells after harmane treatment. ( C ) GSEA illustrating significant activation of the p53 signaling pathway in harmane-treated CRC cells. ( D ) Western blot analysis of p53, its downstream target RRM2B, and apoptosis-related proteins in CRC cells after harmane treatment. ( E, F ) Validation of RRM2B knockdown efficiency using three independent siRNAs via ( E ) qPCR and ( F ) Western blot analysis. Statistical significance was determined using one-way analysis of variance (ANOVA). ( G ) Western blot analysis of apoptosis-related proteins following RRM2B knockdown. ( H ) Cell viability was assessed by CCK-8 assay after RRM2B silencing and harmane treatment in CRC cells. Statistical significance was determined using a t -test. ( I ) Metastatic capabilities were evaluated by transwell migration and invasion assays after RRM2B silencing and harmane treatment in CRC cells. ( J ) After knockdown of endogenous RRM2B, RRM2B or its catalytically inactive mutant (RRM2B Y331F) was overexpressed to investigate its effects on harmane-induced apoptosis in CRC cells. Statistical significance was determined using two-way analysis of variance (ANOVA). ( K, L ) Cell viability and apoptosis were assessed by ( K ) CCK-8 assay and ( L ) flow cytometry to evaluate the effects of overexpression of RRM2B or its catalytically inactive mutant (RRM2B Y331F) on harmane-induced apoptosis in CRC cells following knockdown of endogenous RRM2B. Statistical significance was determined using two-way analysis of variance (ANOVA). All data were derived from one representative experiment out of three independent biological replicates. Results are presented as means ± SEM. *** P < 0.005, **** P < 0.001.

Article Snippet: Following overnight incubation, CRC cells were treated with either 100 μM harmane (MedChemExpress, USA) or a vehicle control for 24 h. After treatment, 10 μL of CCK-8 solution (Vazyme, China) was added directly to each well, and the plates were incubated at 37°C for 4 h. Cell viability was determined at OD at 450 nm using a microplate reader (Thermo Scientific, USA).

Techniques: Quantitative Proteomics, Control, Western Blot, Expressing, Activation Assay, Biomarker Discovery, Knockdown, CCK-8 Assay, Migration, Mutagenesis, Flow Cytometry, Over Expression, Derivative Assay

Journal: mSystems

Article Title: Harmane induces apoptosis through RRM2B and suppresses colorectal cancer progression

doi: 10.1128/msystems.01704-25

Figure Lengend Snippet: Harmane inhibits CRC progression in vivo ( n = 6 per group). ( A ) Experimental timeline illustrating MC38 tumor cell implantation and harmane administration in C57BL/6J mice. ( B ) Representative images of excised mouse tumors from control and harmane-treated groups. ( C ) Body weight records of mice in the control and harmane-treated groups throughout the experiment. Statistical significance was determined using one-way analysis of variance (ANOVA). ( D, E ) Comparisons of ( D ) tumor volume progression and ( E ) final tumor weight in control and harmane-treated mice. Statistical significance was determined using one-way ANOVA ( F, G ) Immunohistochemical analysis of tumor proliferation and apoptosis between control and harmane-treated mice. Statistical significance was determined using two-way ANOVA ( H ) Schematic diagram of fecal microbiota transplantation (FMT). ( I ) Representative images of mouse tumors from the fecal microbiota transplantation control and the harmane-treated group. ( J ) Final tumor weight of mice in the fecal microbiota transplantation control and harmane-treated groups. Statistical significance was determined using a t -test. ( K ) Schematic diagram illustrating that harmane, a differential metabolite identified in the intestines of healthy individuals, advanced adenoma patients, and CRC patients, induces apoptosis in CRC cells through RRM2B-mediated pathways, leading to suppression of tumor growth and progression. Data are derived from one representative experiment out of three independent biological replicates. Results are presented as means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: Following overnight incubation, CRC cells were treated with either 100 μM harmane (MedChemExpress, USA) or a vehicle control for 24 h. After treatment, 10 μL of CCK-8 solution (Vazyme, China) was added directly to each well, and the plates were incubated at 37°C for 4 h. Cell viability was determined at OD at 450 nm using a microplate reader (Thermo Scientific, USA).

Techniques: In Vivo, Control, Immunohistochemical staining, Transplantation Assay, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α + dendritic cells

doi: 10.1084/jem.20100223

Figure Lengend Snippet: XCR1 is functionally active on mouse CD8α + , human BDCA3 + , and sheep CD26 + DCs, and Xcl1 mRNA is stored in quiescent NK cells and memory CD8 + T lymphocytes. Transwell migration assay on enriched human blood DCs or lymphocytes, sheep lymph CD26 + versus CD26 − DCs, and splenic DCs from XCR1 −/− and C57BL/6NCrl (XCR1 +/+ ) mice. Results are representative of at least two independent experiments for each species and expressed as mean ± SEM from duplicate wells for each data point. (B) Expression of the XCL1 gene in human and mouse cell types and tissues, based on the same gene chips data as used in , with the following additions: black triangles, NK cells; purple triangles, resting peripheral CD8 + T cells; purple diamond, anti-CD3 activated human T cells; purple square, mouse CD8 + thymocytes; violet square, mouse CD4 + thymocytes. Results are expressed as mean and SD for at least three independent values for most human data points. (C) Expression of the XCL1 gene in sheep leukocytes as assessed by real-time PCR on the same lymph or blood cells as shown in . Results are mean ± SEM of triplicate real-time RT-PCR reactions, and they are representative of two different sheep for lymphocytes and of three different sheep for DCs. (D) Results of Xcl1 gene expression in mouse CD8 + T cell subsets. Xcl1 expression was measured by real-time PCR on sorted naive or T IM , or antiviral T CM CD8 + T cell subsets. Expression of Ccl5 and Ifng were also evaluated as controls, as the genes are expressed to higher levels in memory CD8 + T cells . Results are represented as mean ± SD for mean values from duplicate real-time PCR reactions performed on mRNAs from naive T cells or from T IM from three individual mice each, and from T CM from four individual pools of seven mice.

Article Snippet: The lower chamber was filled with migration medium alone or containing 300 ng/ml of mouse recombinant XCL1 (R&D Systems) for mouse cells, 500 ng/ml of human recombinant XCL1 (R&D Systems) for human cells, and 100 ng/ml of recombinant mouse XCL1 for sheep cells.

Techniques: Transwell Migration Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Gene Expression

Journal: eLife

Article Title: Cytotoxic T cells swarm by homotypic chemokine signalling

doi: 10.7554/eLife.56554

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant Mouse XCL1 , R & D systems , Cat. #: 486-LT-025 , (1, 10 and 100 ng/ml).

Techniques: In Vivo, Cell Culture, Transduction, Construct, Sequencing, Produced, Transfection, Expressing, Plasmid Preparation, Clone Assay, Control, Recombinant, In Vitro, Mass Spectrometry, Cell Isolation, Selection, Reverse Transcription, Sandwich ELISA, Cytometry, Software