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: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: (a ) Western blotting analysis of CD133, secreted and endogenous CXCL3 proteins expression in HCC cell lines. The gels in the same panel mentioned in this article had been run under the same experimental conditions. Uncropped full-length blots were showed in the . ( b ) CD133 + HCC cells expressed higher level of secreted and endogenous CXCL3 proteins compared with the corresponding CD133 − cells. Uncropped full-length blots were showed in the .

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Western Blot, Expressing

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) MTT assays showed that down-regulation of CXCL3 expression by shRNA inhibited cell growth in Hep3B, Huh7 and PLC/PRF/5 cells after day 4 (values were represented as the mean ± SD; * p < 0.05 vs shNC control, the Bonferroni method was used for the multiple comparison). ( b ) Down-regulation of CXCL3 inhibited cell growth in HCC-LY5, SMMC-7721 and MHCC-LM3 cells after day 4 (values were represented as the mean ± SD; * p < 0.05 vs shNC control, the Bonferroni method was used for the multiple comparison). ( c ) Results of the clone-formation assay in CXCL3 knockdown CD133 + /CD133 − HCC cells sorted from HCC-LY5 ( p = 0.0005) and SMMC-7721 ( p = 0.028) (values were represented as the mean ± SD; * p < 0.05; t -test, vs cells treated with shNC). ( d ) Tumor-sphere formation analysis displayed that CXCL3-silence decreased the tumor-sphere number in Huh7 ( p = 0.0325) and PLC/PRF/5 ( p = 0.0491) CD133 + cells (values were represented as the mean ± SD; * p < 0.05; t -test, vs cells treated with shNC). ( e ) The weight of tumors from BALB/c (nu/nu) mice injected with CXCL3 knockdown or control SMMC-7721 cells are shown (CD133 + -NC vs CD133 − -NC, p = 0.045; CD133 + -NC vs CD133 + -shCXCL3, p = 0.037) (values were represented as the mean ± SD; * p < 0.05 vs cells treated with shNC, the Bonferroni method was used for the multiple comparison).

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Expressing, shRNA, Control, Comparison, Tube Formation Assay, Knockdown, Injection

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) 100 ng/ml exogenous CXCL3 treatment induced Erk1/2 phosphorylation in a time dependent manner in Huh7, HCC-LY5 and SMMC-7721 cells. Uncropped full-length blots were showed in the . ( b ) Long time treatment of CXCL3 (100 ng/ml) induced Erk1/2 and EST1 phosphorylation, and promoted CD133 expression in HCC cells. Uncropped full-length blots were showed in the .

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Phospho-proteomics, Expressing

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) CD133 was overexpressed in HCC-LY5 and SMMC-7721 cells and the luciferase activities associated with CXCL3 promoter are shown. Reporter gene activities are expressed as fold changes relative to the control ( p = 0.002 in HCC-LY5; p = 0.003 in SMMC-7721) (values were represented as the mean ± SD; * p < 0.05; t -test, vs vector control). ( b ) CD133 overexpression up-regulated CXCL3 protein expression in HCC cell lines. Uncropped full-length blots were showed in the . ( c ) CD133 knockdown down-regulated CXCL3 expression in HCC cells. Uncropped full-length blots were showed in the .

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Luciferase, Control, Plasmid Preparation, Over Expression, Expressing, Knockdown

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: ( a ) Realtime PCR analysis demonstrated that HCC tumor tissue samples expressed higher level of CXCL3 mRNA (22/30) compared with the para-tumor tissue samples. ( b ) Western blotting analysis of 7 pair tissue samples showed that CXCL3 HCC tumor tissue samples (T) expressed higher level of CXCL3 protein compared with the para-tumor tissue samples (N). Uncropped full-length blots were showed in the . ( c ) ELISA results displayed that serum CXCL3 lever was higher in HCC patients ( p = 0.0418)(values were represented as the mean ± SD; ** p < 0.01; t -test, vs normal people control). ( d ) The cutoff value of 66.36 pg/ml for serum CXCL3 with a sensitivity of 62.4% and specificity of 88.1% using receiver operating characteristic analysis. ( e ) Results of overall survival analysis of serum CXCL3 in HCC patients are shown (Kaplan-Meier analysis).

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: CXCL3 contributes to CD133 + CSCs maintenance and forms a positive feedback regulation loop with CD133 in HCC via Erk1/2 phosphorylation

doi: 10.1038/srep27426

Figure Lengend Snippet: Correlation between Clinicopathologic Features and Serum CXCL3 Protein Expression.

Article Snippet: ELISA was performed with the Human C-X-C motif chemokine 3 (CXCL3) ELISA Kit according to the manufacturer’s instructions (CUSABIO, Wuhan, Hubei, China).

Techniques: Expressing

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: The Journal of investigative dermatology

Article Title: Profiling Motility Signal-Specific Genes in Primary Human Keratinocytes

doi: 10.1038/jid.2008.34

Figure Lengend Snippet: HKs, infected with FG12 system carrying vector alone, control siLac-Z, or small interfering RNA against each of the three candidate genes, were subjected to RT-PCR, immunoblotting, and colloidal gold migration assays. (a) Total RNA from these cells was isolated 5 days following infection and was subjected to RT-PCR. (b–f) To detect the protein expression levels, lysates of the HKs, starved in serum-free medium overnight, were extracted and immunoblotted with antibodies against HB-EGF or CXCL3. To detect secreted MMP-10 proteins, infected HKs were starved in serum-free medium overnight. Conditioned medium (CM) was collected and concentrated 100 times. The CM was analyzed by western blot with an anti-MMP-10 antibody (f). (g) After the effectiveness of the RNAi was confirmed, colloidal gold migration assay was carried out to functionally characterize the importance of these genes in HK migration. The experiments were repeated three times (*P<0.05).

Article Snippet: The anti-human CXCL3 antibody was from Aviva Systems Biology (San Diego, CA).

Techniques: Infection, Plasmid Preparation, Control, Multiplex sample analysis, Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Migration, Isolation, Expressing

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