Journal: eLife

Article Title: A non-bactericidal cathelicidin provides prophylactic efficacy against bacterial infection by driving phagocyte influx

doi: 10.7554/eLife.72849

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse CXCL3 ELISA Kit , Rockland , Cat#:KOA0825 , .

Techniques: Flow Cytometry, cDNA Library Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Staining, Blocking Assay, In Vivo, Control

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: ( A ) α Diversity analysis using the Chao1 and the Simpson indexes in the CRC ( n = 20) and HC ( n = 20) groups. ( B ) Principal coordinate analysis at the species level between the CRC ( n = 20) and HC ( n = 20) groups. ( C ) The discriminant analysis effect size method identified marker species between the CRC ( n = 20) and HC ( n = 20) groups. Blue and red bars represent markers enriched in the CRC and HC groups, respectively. ( D ) A human cytokine antibody array was applied to detect the changes of inflammatory factors in CM of HCT116 cells treated with F . nucleatum or E. coli . Differential cytokines associated with immune cell chemotaxis are shown in the black boxes. A cytokine chip Wayne diagram is shown below those boxes. ( E and F ) qPCR analysis revealed changes in cytokine expression after co-culture with F . nucleatum and E. coli for 6 hours ( n = 3). ( G ) ELISA detection of CCL20 and CXCL3 secretion from CRC cells after co-culture with E. coli and F. nucleatum ( n = 5). All numerical data and error bars represent the mean ± SD of 3 independent experiments. Statistical analyses were conducted using 1-way ANOVA with Dunnett’s T3 correct multiple-comparison test. * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques: Marker, Ab Array, Chemotaxis Assay, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: ( A ) Knockdown of Ccl20 and Cxcl3 inhibits the growth of CT26 subcutaneous tumors in nude mice ( n = 5). A photograph of CT26 subcutaneous tumors in nude mice and a graph of tumor growth are shown. ( B ) Knockdown of Ccl20 and Cxcl3 inhibits the growth of CT26 subcutaneous tumors in BALB/c mice ( n = 5). A photograph of CT26 subcutaneous tumors in BALB/c mice and a graph of tumor growth are shown. ( C ) Neutralizing antibodies of Ccl20 and Cxcl3 inhibited subcutaneous tumors in BALB/c mice( n = 6). A photograph of CT26 subcutaneous tumors in BALB/c mice and a graph of tumor growth are shown. ( D ) Knockdown of Ccl20 and Cxcl3 inhibits the progression of CT26 orthotopic implanted tumor in BALB/c mice ( n = 5). Representative tumor images and tumor load are shown. ( E ) The percentage of PMN-MDSCs (CD11b + Ly6G + Ly6C low ) in TILs (CD45 + ) of orthotopic implanted CRC mice detected by flow cytometry sorting. A bar chart indicating statistical values is presented ( n = 5). ( F ) The percentage of Tregs (CD4 + Foxp3 + ) in TILs (CD45 + ) of orthotopic implanted CRC mice detected by flow cytometry sorting. A bar chart indicating statistical values is presented ( n = 5). ( G and H ) Tumor-infiltrating CD8 + T cells and their granule production (GzmB + ) in TILs (CD45 + ) of orthotopic implanted CRC mice detected by flow cytometry sorting. Bar charts indicating statistical values are presented ( n = 5). ( I ) MDSCs (Gr-1 + ), Tregs (CD4 + , Foxp3 + ), and CD8 + T-cell infiltration in tumor tissues of orthotopic implanted CRC mice. Representative IHC images are shown. ( J ) Histogram showing the number of Gr-1 + , CD4 + , Foxp3 + , and CD8 + cells per ×20 objective lens visual field ( n = 5). Data represent the mean ± SD of 3 independent experiments. We used 2-way ANOVA to determine statistical significance of subcutaneous tumor volume. The remaining statistical methods were conducted using 1-way ANOVA with Dunnett’s T3 correct multiple-comparison test. * P < 0.05, ** P < 0.005, *** P < 0.0005. sh, short hairpin.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques: Knockdown, Flow Cytometry, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: ( A ) IF assays were performed to detect CXCR2 and Gr-1 in orthotopic cecal tumor of BALB/c mice. Scale bar: 50 μm. ( B and C ) The CXCL3-CXCR2 axis promoted the migratory abilities of MDSCs, as detected by transwell assays ( n = 5). ( D and E ) Representative flow cytometry data show that MDSCs cells isolated from C57 mice inhibited cytokine and cytolytic granule production in CD8 + T cells ( D ); the summarized result is presented in ( E ) ( n = 5). ( F ) Effect of short hairpin Cxcl3 (sh- Cxcl3 ) and PD-1 immunotherapy on subcutaneous tumor of BALB/c mouse: CT26 subcutaneous tumors ( n = 5). The CD279 anti–PD-1 antibody or isotype control (IgG) was i.p. injected three times daily ( G and H ) Tumor growth ( G ) and weight ( H ) were monitored ( n = 5). Data represent the mean ± SD of 3 independent experiments. Statistical analyses were conducted using Student’s t test (2-comparison test) and 1-way ANOVA with Dunnett’s T3 correct multiple-comparison test. We used 2-way ANOVA to determine statistical significance of tumor volume. * P < 0.05; ** P < 0.005; *** P < 0.0005.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques: Flow Cytometry, Isolation, Control, Injection, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: ( A ) Identification of differential metabolites in the E . coli and F . nucleatum imbalance models using nontargeted metabolomics, presented volcano plots. ( B ) Principal component analysis (PCA) comparing the E . coli or F . nucleatum imbalance groups with the control group. ( C ) A Wayne chart illustrating differential metabolites between the E . coli or F . nucleatum imbalance models and the control group. ( D and E ) Heatmaps depicting differential metabolites in the E . coli ( D ) and F . nucleatum ( E ) imbalance groups compared with the control groups. P < 0.05, 2-tailed Mann-Whitney U test. ( F and G ) ELISA was used to assess the impact of differential metabolites (1 mM; 48 hours) on CXCL3 levels ( n = 5). ( H – K ) ELISA assays measuring the effects of the 4-HPA concentration gradient and time gradient on CXCL3 secretion ( n = 5). Data represent the mean ± SD of 3 independent experiments. Statistical analyses were conducted using 1-way ANOVA with Dunnett’s T3 correct multiple-comparison test. * P < 0.05, *** P < 0.0005. PC2, principal components 2; QC, quality control; VIP, variable importance in projection.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques: Control, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: ( A ) Chemical structures of 4-HPA, FITC, and FITC-labeled -HPA. ( B ) Detection of FITC-labeled 4-HPA by fluorescence confocal microscopy in RKO and SW480 cells. Scale bar: 50 μm. ( C ) Predicted transcription factors for CXCL3. ( D ) WB analysis of the effects of 4-HPA on transcription factors in CRC cell lines. ( E ) The binding sites of STAT3 and CXCL3 were confirmed using a dual-luciferase reporter assay ( n = 3). ( F ) Transcriptional regulation of CXCL3 by p-STAT3 was detected using ChIP assays. ( G ) WB analysis of the JAK2/STAT3 signaling pathway and CXCL3 in SW480 cells. ( H – J ) WB analysis of CXCL3 in SW480 cells. ( K and L ) IF assays visualizing the subcellular localization of STAT3 and p-STAT3 in SW480 cells treated with 4-HPA. Scale bar: 50 μm. Data represent the mean ± SD of 3 independent experiments. Statistical analyses were conducted using Student’s t test. *** P < 0.0005. MUT, mutation; NC, negative control.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques: Labeling, Fluorescence, Confocal Microscopy, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: ( A ) Tumors in the intestines of Apc min/+ tumorigenesis mice ( n = 5). ( B ). Bar chart of tumor load in intestines derived from Apc min/+ mice treated with PD-1 immunotherapy or IgG, with or without 4-HPA (1 mM) ( n = 5). ( C ) Representative images of intestines tumorigenesis visualized by H&E staining. ( D ) H&E scoring of tumor-related lesions (including inflammation, adenoma, atypical hyperplasia, and crypt fusion) ( n = 5). ( E and F ) Detection of 4-HPA in tumor tissues of patients with CRC by HRGC-MS ( n = 12). ( G – K ) Expression of CD8, CD33, CD11b, and CXCL3 in CRC tissues of patients with CRC analyzed by multiple IF. Visualization of 3 representative cases is shown. Scale bar: 50 μm. Multiple IF detection was performed on the tumor tissues of patients with CRC ( G ). Bar charts of CD8 + T cells ( H ), CD11b + cells ( I ), CD33 + cells ( J ), and MDSCs (CD11b + CD33 + ) ( K ) in CRC tissues ( n = 5). Data represent the mean ± SD of 3 independent experiments. We used 2-way ANOVA to determine the significance of tumor volume of PD-1 treated mice and H&E staining. The remaining statistical analyses were conducted with Student’s t test (2-comparison test) and 1-way ANOVA with Dunnett’s T3 correct multiple-comparison test. * P < 0.05, ** P < 0.005, *** P < 0.0005. rel., relative.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques: Derivative Assay, Staining, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Gut microbial metabolite 4-hydroxybenzeneacetic acid drives colorectal cancer progression via accumulation of immunosuppressive PMN-MDSCs

doi: 10.1172/JCI181243

Figure Lengend Snippet: The gut microbiome uses 4-HPA as a messenger to regulate chemokine CXCL3 level in CRC cells, thereby controlling the accumulation of CXCR2 + PMN-MDSCs. The accumulated PMN-MDSCs inhibit the antitumor effect of CD8 + T cells.

Article Snippet: Cytokine neutralization was performed with anti- Cxcl3 monoclonal antibody (sheep; R&D Systems, AF5568), anti- Ccl20 monoclonal antibody (rabbit; R&D Systems, MAB7601), and IgG isotype control (BioXCell, BE0090, clone 2A3) given by i.p. injection twice a week at a dose of 200 μg/injection.

Techniques:

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: Intracerebellar treatment with Cxcl3 dramatically reduces lesion volume in 4‐month‐old Ptch1 + / − / Tis21 − / − mice. (A) Treatment timeline: 3‐month‐old Ptch1 + / − / Tis21 − / − mice were treated for 28 days with Alzet minipumps filled with recombinant Cxcl3 or vehicle (CSF); a single injection of BrdU was performed 5 days before immunohistochemical analysis. (B) MB lesion frequency and number of preneoplastic lesions per cerebellum in CSF‐treated and Cxcl3‐treated mice. The number of mice analyzed for each treatment and the statistical analysis are indicated. (C) Representative confocal images of cerebellar sagittal slices of 4‐month‐old Ptch1 + / − / Tis21 − / − mice treated with CSF (left) or Cxcl3 (right), respectively. Nuclei were stained with Hoechst 33258 and lesions were identified by the presence of BrdU + pGCPs (red). Scale bar, 500 μm. (D, E) Bar graphs show the mean ± SEM of lesion areas (D) and lesion volumes (E) measured at the end of 28 days of treatment with Cxcl3 or vehicle. * p < 0.05, **** p < 0.0001, Student's t test; mice analyzed: n = 8 for vehicle, n = 9 for Cxcl3.

Article Snippet: The Alzet minipump (1004, which delivers 0.11 μl/h for 4 weeks; Durect Corp.) was filled with recombinant Cxcl3 (100 μl of a solution at 20 μg/ml; 5568‐CA‐025/CF, R&D Systems, Minneapolis, MN, USA) or with the vehicle (cerebrospinal fluid [CSF] solution, as per Durect Corp. protocol: 148 mM NaCl, 3 mM KCl, 1.4 mM CaCl 2 , 0.8 mM MgCl 2 , 8 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 ) and placed between the scapulas.

Techniques: Recombinant, Injection, Immunohistochemical staining, Staining

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: In 4‐month‐old Ptch1 + / − / Tis21 − / − mice, treatment with Cxcl3 induces neoplastic precursors to migrate from MB lesions to the IGL and differentiate. (A) Experimental timeline: 3‐month‐old Ptch1 + / − / Tis21 − / − mice were treated for 4 weeks with Alzet minipumps filled with recombinant Cxcl3 or CSF alone; mice received a single injection of BrdU at P113 and were analyzed by immunohistochemistry at P118. (B) Representative confocal images of pGCPs migrating out of MB lesions, identified as BrdU + cells (red), in Ptch1 + / − / Tis21 − / − cerebella chronically treated with CSF or Cxcl3. Sections are counterstained with Hoechst 33258 to visualize the lesion (L), molecular layer (ML), and internal granular layer (IGL). Scale bar, 50 μm. (C) Migrating pGCPs were quantified as percentage ratio (mean ± SEM) of BrdU + cells present within the ML or IGL area adjacent to each lesion to the total number of BrdU + cells in the lesion, ML and IGL. ** p < 0.01, **** p < 0.0001, Student's t test; mice analyzed: n = 6 for each treatment. (D) The same confocal sections of (B) were also reacted with antibody against NeuN (green), to label migrating and differentiating pGCPs (BrdU + NeuN + ). Sections are counterstained with Hoechst 33258 to visualize the lesion, ML and IGL. Scale bar, 50 μm. Some differentiated cells in the IGL are indicated by white arrows. (E) Quantification of the percentage ratio (mean ± SEM) of BrdU + NeuN + cells present within the ML or IGL area adjacent to each lesion to the total number of BrdU + cells in the lesion, ML and IGL. * p < 0.05, **** p < 0.0001, Student's t test; mice analyzed: n = 6 for each treatment.

Article Snippet: The Alzet minipump (1004, which delivers 0.11 μl/h for 4 weeks; Durect Corp.) was filled with recombinant Cxcl3 (100 μl of a solution at 20 μg/ml; 5568‐CA‐025/CF, R&D Systems, Minneapolis, MN, USA) or with the vehicle (cerebrospinal fluid [CSF] solution, as per Durect Corp. protocol: 148 mM NaCl, 3 mM KCl, 1.4 mM CaCl 2 , 0.8 mM MgCl 2 , 8 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 ) and placed between the scapulas.

Techniques: Recombinant, Injection, Immunohistochemistry

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: Expression of the chemokine receptor Cxcr2 in MB tissue of Ptch1 + / − / Tis21 − / − mice and DAOY cells. (A) Representative confocal images of Cxcr2 + cells (red) in MB lesion (left) and tumor tissue (middle) of Ptch1 + / − / Tis21 − / − mice, and SHH‐type MB cell line DAOY (right). Sections are counterstained with Hoechst 33258 to visualize nuclei. Scale bar, 50 μm. Cells in boxed areas are shown at higher digital magnification (3×). (B) Test of the ability to migrate in response to Cxcl3 of DAOY cells by scratch wound healing assay. Representative photomicrographs of DAOY cells (magnification 4×; scale bar 500 μm) taken at time of scraping (0 h) and 24 h after treatment with 0.1% BSA (as control; top) or Cxcl3 (bottom). Dashed white lines indicate wound boundaries. (C) Mean ± SEM of migration rate calculated as reported in the Materials and Methods section 24 h after wound scratching in control or Cxcl3‐treated DAOY cells. (D) Quantification of migration rate, expressed as mean ± SEM, 24 and 48 h after wound scratching in DAOY cells treated with vehicle alone (white bars), with the chemokine Cxcl3 (black bars), with Reparixin (Rpx, red bars), or with the combination Cxcl3 + Rpx (blue bars), respectively. (C, D) Data were obtained from two separate experiments, counting approximately four fields per well (at least three wells per experiment). * p < 0.05, *** p < 0.001, Student's t test.

Article Snippet: The Alzet minipump (1004, which delivers 0.11 μl/h for 4 weeks; Durect Corp.) was filled with recombinant Cxcl3 (100 μl of a solution at 20 μg/ml; 5568‐CA‐025/CF, R&D Systems, Minneapolis, MN, USA) or with the vehicle (cerebrospinal fluid [CSF] solution, as per Durect Corp. protocol: 148 mM NaCl, 3 mM KCl, 1.4 mM CaCl 2 , 0.8 mM MgCl 2 , 8 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 ) and placed between the scapulas.

Techniques: Expressing, Wound Healing Assay, Control, Migration

Journal: Brain Pathology

Article Title: Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells

doi: 10.1111/bpa.13283

Figure Lengend Snippet: In vivo imaging of an orthotopic xenograft mouse model obtained by intracerebellar implantation of engineered human DAOY MB cells. (A) Treatment timeline: Six/seven‐week‐old athymic nude mice ( Foxn1 nu / Foxn1 + ) were implanted into the left cerebellar hemisphere with 1 × 10 5 human DAOY MB cells engineered to express GFP and firefly Luciferase (FLuc) genes (D0); 14 days after cell grafting (D14), mice were grouped according to their bioluminescence values and implanted with Alzet minipumps filled with recombinant Cxcl3 or CSF. During 4 weeks of treatment, mice were imaged every 7 days (D21, 28, 35, and 42) via in vivo bioluminescent imaging to monitor tumor growth and spinal cord metastases. (B) Left: DAOY cells, infected with a lentiviral vector encoding GFP‐FLuc, express GFP in vitro as determined by confocal fluorescence imaging (green cells; nuclei were stained with Hoechst 33258). Middle: DAOY cells implanted in the cerebellum of nude mice give rise to large intracerebellar tumors, as visible by illuminating the explanted brains at the end of treatment with a “GFP flashlight” (Nightsea) which causes GFP + tumors to glow. Right: Representative image by confocal microscopy of a cerebellar sagittal slice obtained from the brain shown in the middle image, showing MB cells, identified as GFP + cells (in green), 42 days after intracerebellar injection of engineered DAOY cells. The section is counterstained with Hoechst 33258 to visualize the ML and granule neurons in the IGL. Scale bars, 100 μm (left), 1 cm (middle), and 600 μm (right). (C) Representative bioluminescence images of nude mice orthotopically injected with DAOY‐Luc MB cells and after 14 days implanted with Alzet minipumps filled with CSF or Cxcl3, respectively (7 mice for each treatment group). Images are shown for days 21, 28, 35, and 42 after injection of DAOY‐Luc cells. Three animals, one CSF‐treated mouse (#5) and two Cxcl3‐treated mice (#11 and #14), developed metastases during the 28 days of Alzet implantation. (D) Tumor growth according to quantified electron emission (e − /s) from the cerebellar region of mice injected with DAOY‐Luc cells and treated in vivo for 4 weeks with Cxcl3 (orange) or CSF as vehicle (blue). The filled circles and the error bars indicate the mean ± SEM of the bioluminescence imaging (BLI) increase measured at the days of treatment indicated with respect to the day of Alzet implantation (D14). p = 0.4124 at D21, p = 0.8462 at D28, p = 0.9867 at D35, p = 0.9500 at D42, Student's t test.

Article Snippet: The Alzet minipump (1004, which delivers 0.11 μl/h for 4 weeks; Durect Corp.) was filled with recombinant Cxcl3 (100 μl of a solution at 20 μg/ml; 5568‐CA‐025/CF, R&D Systems, Minneapolis, MN, USA) or with the vehicle (cerebrospinal fluid [CSF] solution, as per Durect Corp. protocol: 148 mM NaCl, 3 mM KCl, 1.4 mM CaCl 2 , 0.8 mM MgCl 2 , 8 mM Na 2 HPO 4 , 0.2 mM NaH 2 PO 4 ) and placed between the scapulas.

Techniques: In Vivo Imaging, Luciferase, Recombinant, In Vivo, Imaging, Infection, Plasmid Preparation, In Vitro, Fluorescence, Staining, Confocal Microscopy, Injection

Journal: Nature Communications

Article Title: ARID1A loss induces polymorphonuclear myeloid-derived suppressor cell chemotaxis and promotes prostate cancer progression

doi: 10.1038/s41467-022-34871-9

Figure Lengend Snippet: a Volcano plots showing the differentially expressed genes (DEGs) between epithelial cells of 3-month-old Pten PC −/− ; Arid1a PC −/− versus Pten PC −/− mouse prostates ( n = 3). The upregulated and downregulated cytokines and chemokines are indicated. p -value was determined by DEGseq analysis. b IB analysis of the indicated protein in 3-month-old mouse prostates. c IB analysis of WT and ARID1A -depleted C4-2 and Myc-CaP cells treated with TNFα at the indicated time points. d IB analysis in WT and ARID1A -overexpression Myc-CaP cells with or without TNFα stimulation. e Heatmap summarizing the qRT-PCR results in WT and Arid1a KO cells with or without TNFα stimulation. f ELISA of CXCL2 and CXCL3 in serum and prostate tumors of 3-month-old mice ( n = 5). g Tumor volume of Myc-CaP expressing sg ARID1A or control vector subcutaneously inoculated into FVB mice with or without JSH-23 treatment (WT + DMSO, n = 5; Arid1a KO + DMSO, n = 7; WT/ Arid1a KO + JSH-23, n = 6). h Epithelial cells in xenografts ( g ) were sorted for RNA-Seq and DEGs between WT and Arid1a KO epithelium were shown in heatmap ( n = 2). i Migration of PMN-MDSCs recruited by conditional mediums (CMs) with the indicated treatments ( n = 3). j , k Mice were inoculated with WT or Arid1a KO Myc-CaP cells and treated with or without SB225002. Tumor volume was monitored ( j , WT + DMSO, n = 5; Arid1a KO + DMSO, n = 7; WT/ Arid1a KO + JSH-23, n = 6) and quantification of each tumor-infiltrating immune cell population ( k , n = 5) were measured by FACS analysis. f , g , i – k Data represent the mean ± SEM. Statistical significance was determined by two-tailed unpaired t -test ( f , i , k ) and two-way ANOVA followed by multiple comparisons ( g , j ). b – d Data were evaluated in triplicate, and representative data are shown. Source data are provided as a Source Data file. ns, no significance.

Article Snippet: The amount of CXCL2 and CXCL3 protein in the serum and protein lysis of prostate tissues from Pten PC −/− and Pten PC −/− ; Arid1a PC −/− mice was determined using mouse CXCL2 and CXCL3 specific ELISA kits (Elabscience; E-EL-M0019c and Beijing BioRab Technology Co. Ltd.; ZN2584).

Techniques: Over Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Control, Plasmid Preparation, RNA Sequencing, Migration, Two Tailed Test

Journal: Nature Communications

Article Title: ARID1A loss induces polymorphonuclear myeloid-derived suppressor cell chemotaxis and promotes prostate cancer progression

doi: 10.1038/s41467-022-34871-9

Figure Lengend Snippet: a Representative IB results of ARID1A, p-IKKβ, p-P65 and A20 expression in the lysates of human prostate tumors. Pearson’s correlations among proteins indicated in PCa specimens are summarized in the heatmap ( n = 42; GS > 7). b ELISA of CXCL2 and CXCL3 in PCa ( n = 42). c Heatmap summary of the correlations of the indicated signature in PCa ( n = 150, GSE21032; n = 266, Prad_SU2C_2019). d IHC analysis for ARID1A, P65, CD15 and CD8 markers. Scale bars, 50 μm. The correlations between ARID1A expression and nuclear P65 intensity and the abundance of CD15 + and CD8 + cells are shown as stacked columns ( n = 100). e Volume of tumors derived from WT and ARID1A -overexpressing cells injected subcutaneously into FVB mice and treated with IgG and anti-PD1 antibody ( n = 10). f Prostate tumor histology of Pten PC −/− ; Arid1a PC −/ mice with or without NF-κB inhibition (JSH-23) in combination with anti-PD1/CTLA-4 treatment ( n = 10), Scale bars, 50 μm. g IHC staining for Ki67, CD8 and Ly6G and β-Gal in sections by the indicated treatments. Scale bars, 50 μm. h ARID1A functions downstream of inflammation-induced IKKβ activation to shape the immunosuppressive TME through the regulation of NF-κB-mediated chemotaxis. b and e Data represent the mean ± SEM. Statistical significance was determined by two-tailed Pearson’s correlations test ( a and c ), two-tailed unpaired t -test ( b ), two-tailed χ 2 ( d ) and two-way ANOVA followed by multiple comparisons ( e ). g Experiments were repeated at least three times independently with similar results; data from one representative experiment are shown. Source data are provided as a Source Data file.

Article Snippet: The amount of CXCL2 and CXCL3 protein in the serum and protein lysis of prostate tissues from Pten PC −/− and Pten PC −/− ; Arid1a PC −/− mice was determined using mouse CXCL2 and CXCL3 specific ELISA kits (Elabscience; E-EL-M0019c and Beijing BioRab Technology Co. Ltd.; ZN2584).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Injection, Inhibition, Immunohistochemistry, Activation Assay, Chemotaxis Assay, Two Tailed Test

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet: CAFs-derived CXCL3 acted as a potential mediator in RCC progression (A) Cell viability% of Renca cells cultured with heated CAFs-CM. (B) The cytotoxicity of sunitinib at different concentrations on Renca co-cultured with heated CAFs-CM. (C) Crystal violet staining and quantification of migrated and invasive Renca cells after 36 h treatment. (D) Proteomics sequencing clustering analysis of mouse-derived CAFs serum-free conditioned medium (CAFs) and NIH-3T3 serum-free conditioned medium (NFs). (E) Differential protein volcano map of CAFs versus NFs serum-free conditioned medium. (F) CXCL3 concentration in the CAFs and NFs conditioned medium. (G) Gene expression of Cxcl3 in CAFs and NFs. (H) Gene expression of CXCL3 in RCC tissue. (I) Protein expression of CXCL3 in tumor tissue of tumor-bearing mice. Scale bar: 100 μm. Data are shown as mean ± SD in (D) and (E), mean ± SEM in other panels, n = 3∼6, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns: no significance.

Article Snippet: CXCL3 enzyme-linked immunosorbent assay kit , Elabscience , Cat# E-EL-M0147c.

Techniques: Derivative Assay, Cell Culture, Staining, Sequencing, Concentration Assay, Gene Expression, Expressing

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet: CAFs promoted RCC progression and sunitinib resistance through CXCL3/CXCR2 (A) Renca mean ± cell viability% of Renca cells was evaluated by the CCK8 assay after stimulation with different doses of CXCL3 (0.5, 1, 2, 5, 10, 20, and 50 ng/mL) for 24 h. (B) Crystal violet staining and quantification of migrated Renca cells after CXCL3 treatment for 36 h. (C) Sunitinib cytotoxicity at a concentration of 20 μM in Renca cells with or without CXCL3 stimulation. (D) Cell viability of Renca cells co-cultured with conditioned medium from CAFs with CXCL3 interference. (E) Transwell migration or invasion and their quantifications of Renca cells stimulated by CXCL3-interfering CAFs-CM. (F) Sunitinib (20 μM) cytotoxicity of Renca cells co-cultured with CAFs-CM-siCXCL3. (G and H) Cell viability and sunitinib cytotoxicity of Renca cells co-cultured with conditioned medium from CXCL3 overexpressed NIH3T3cells. (I–K) The effects of SB225002 on the proliferation, migration and sunitinib resistance of Renca cells induced by CAFs-CM. Scale bar: 100 μm. Data are shown as mean ± SEM, n = 3∼6, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ns: no significance.

Article Snippet: CXCL3 enzyme-linked immunosorbent assay kit , Elabscience , Cat# E-EL-M0147c.

Techniques: CCK-8 Assay, Staining, Concentration Assay, Cell Culture, Migration

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet: CAFs-CM and CXCL3 regulated ERK1/2 signaling pathway (A and B) Western blot analysis and quantification of ERK1/2 and phosphorylated-ERK1/2 expression in Renca cells stimulated with CAFs-CM or CXCL3. (C) Expression of ERK1/2 and phosphorylated-ERK1/2 in Renca cells stimulated with conditioned medium from CAFs with CXCL3 interference. (D) Protein expression of ERK1/2 and phosphorylated-ERK1/2 in tumor tissue of tumor-bearing mice. (E and F) Protein expression of N-cadherin and CD133 in Renca cells stimulated with CAFs-CM or CXCL3. (G) Protein expression of CD133 and E-cadherin in tumor tissue of tumor-bearing mice. Data are shown as mean ± SEM, n = 3∼6, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: CXCL3 enzyme-linked immunosorbent assay kit , Elabscience , Cat# E-EL-M0147c.

Techniques: Western Blot, Expressing

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet: Tumor cells promoted the transformation of NFs into CAFs (A) The gene expression of α-Sma , Fsp1 , and vimentin in NIH3T3cells stimulated by Renca-CM. (B) The gene expression of Fap in MEF stimulated by Renca-CM. (C and D) The gene expression of Cxcl3 in NIH-3T3 and MEF cells treated with Renca-CM. (E) The gene expression of α-Sma , Fap , and vimentin in CAFs stimulated by Renca-CM. (F) The gene expression of Cxcl3 in CAFs treated with Renca-CM. (G and H) Cell viability and sunitinib cytotoxicity of Renca cells co-cultured with conditioned medium from transformed NIH3T3cells. (I and J) Cell viability and sunitinib cytotoxicity of Renca cells co-cultured with conditioned medium from transformed MEF cells. (K–M) Tumor morphology, tumor volume curve, and tumor weight in tumor bearing mice co-transplanted with transformed MEF. (N) Representative morphology and H&E staining of lung tissues co-injected with transformed MEF. Scale bar: 100 μm. Data are shown as mean ± SEM in experiments, n = 3∼5, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: CXCL3 enzyme-linked immunosorbent assay kit , Elabscience , Cat# E-EL-M0147c.

Techniques: Transformation Assay, Gene Expression, Cell Culture, Staining, Injection

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet: Renca cells promoted the transformation and CXCL3 expression of fibroblasts through TGF-β-Smad2/3 signaling pathway (A and B) The gene expression and extracellular secretion of TGF-β in Renca cells following treatment with CAFs-CM. (C) The gene expression of Tgf-β in Renca cells treated by CXCL3. (D and E) The gene expression and extracellular secretion of TGF-β in Renca cells interfered with siRNA. (F and G) The Cxcl3 expression in NIH3T3cells or CAFs co-cultured with conditioned medium from Renca cells with TGF-β interference, respectively. (H and I) The protein expression of Smad2/3, and phosphorylated-Smad2/3 in NIH3T3cells treated by TGF-β and Renca-CM, along with their quantification. Data are shown as mean ± SEM, n = 3∼5, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: CXCL3 enzyme-linked immunosorbent assay kit , Elabscience , Cat# E-EL-M0147c.

Techniques: Transformation Assay, Expressing, Gene Expression, Cell Culture

Journal: iScience

Article Title: CXCL3/TGF-β-mediated crosstalk between CAFs and tumor cells augments RCC progression and sunitinib resistance

doi: 10.1016/j.isci.2024.110224

Figure Lengend Snippet:

Article Snippet: CXCL3 enzyme-linked immunosorbent assay kit , Elabscience , Cat# E-EL-M0147c.

Techniques: Recombinant, Cell Culture, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, RNA Extraction, Sequencing, Over Expression, Plasmid Preparation, Software