Journal: PLoS ONE

Article Title: MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

doi: 10.1371/journal.pone.0070123

Figure Lengend Snippet: A. SQRT-PCR shows increased matrix metalloproteinase mRNA expression in EBS-DM cell lines (n = 4). B. MMP-9 ELISA of 48-h-conditioned cell culture supernatant shows 2-fold upregulation of MMP-9 in KEB-7 and 46-fold upregulation in EBDM-1 at the protein level (n = 4). C. MMP-9 levels were highly increased in EBS patients blister fluids compared to healthy controls (n = 3 to n = 4). The numbers correlate with . Student`s t -test was performed with p values: * ≤0.05, ** ≤0.01, *** ≤0.005, ΔΔ≤0.0005, ΔΔΔ≤0.0001. (In 2C, the Student’s t -test compared the entire patient group to the entire control group).

Article Snippet: Protein levels of KLK5, MMP7, MMP9, CXCL1, CXCL8/IL-8, CXCL11 and CXCL14 were determined in 48-h-conditioned cell culture supernatant and in patients blister fluids by using Quantikine® ELISA (Human KLK5, # DKK500, R&D Systems; Human MMP7, # DMP700, R&D Systems; Human MMP9, # DMP900, R&D Systems; Human CXCL1/GROα, # DGR00, R&D Systems; Human CXCL8/IL-8, # D8000C, R&D Systems; Human CXCL11/I-TAC, # DCX110, Human CXCL14/BRAK, # DY866, R&D Systems) following the manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: PLoS ONE

Article Title: MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

doi: 10.1371/journal.pone.0070123

Figure Lengend Snippet: A. SQRT-PCR of chemokine mRNA expression in NEB-1, KEB-7 and EBDM-1 (n = 3 to n = 5). CXCL1 , CXCL8/IL-8 and CXCL14 expression was increased in KEB-7. Only CXCL11 and CXCL14 were increased in EBDM-1. B. CXCL8/IL-8 ELISA of 48-h-conditioned cell culture supernatant showed 2-fold upregulation at the protein level in KEB-7 but not in EBDM-1, which correlates with the SQRT-PCR results (n = 4). C. CXCL8/IL-8 concentrations were highly increased in EBS patients blister fluids. In blister fluids of healthy controls no CXCL8/IL-8 was detectable (n = 3 to n = 4). The numbers correlate with . Student’s t -test was performed with p values: * ≤0.05, *** ≤0.005, Δ≤0.001, ‡ = no significant difference between investigated cell lines. (In 6C, the Student’s t -test compared the entire patient group to the entire control group).

Article Snippet: Protein levels of KLK5, MMP7, MMP9, CXCL1, CXCL8/IL-8, CXCL11 and CXCL14 were determined in 48-h-conditioned cell culture supernatant and in patients blister fluids by using Quantikine® ELISA (Human KLK5, # DKK500, R&D Systems; Human MMP7, # DMP700, R&D Systems; Human MMP9, # DMP900, R&D Systems; Human CXCL1/GROα, # DGR00, R&D Systems; Human CXCL8/IL-8, # D8000C, R&D Systems; Human CXCL11/I-TAC, # DCX110, Human CXCL14/BRAK, # DY866, R&D Systems) following the manufacturer’s protocol.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Control

Journal: NPJ Regenerative Medicine

Article Title: Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal

doi: 10.1038/npjregenmed.2016.17

Figure Lengend Snippet: Cxcl14 is expressed in muscle cells. ( a ) C2C12 cell media over the course of differentiation (0, 24, 48, 72 h) were subjected to ELISA assay to determine secreted Cxcl14 levels ( n =5, samples assayed in duplicates). ( b ) TA muscles were injured by BaCl 2 injection and isolated on days 3, 5, 7 and 14 after injury (AI). Upon cryosection, H&E staining or immunofluorescence staining for Cxcl14 (green) together with DAPI (blue) was performed ( n =3). Scale bars: 50 μm. ( c ) The procedure described in ( b ) was repeated. Muscle sections isolated on day 3 AI were immunofluorescently probed for MyoD (red), Cxcl14 (green) and DAPI (blue) ( n =6). Scale bar: 15 μm. Paired two-tailed t -test was performed for the data in ( a ). The data denoted by different letters ( a – c ) are significantly different from each other ( P <0.05). All error bars represent s.d. of independent replicates.

Article Snippet: U0126 and ELISA kit for detection of mouse Cxcl14 was from Thermo Scientific.

Techniques: Enzyme-linked Immunosorbent Assay, Muscles, Injection, Isolation, Staining, Immunofluorescence, Two Tailed Test

Journal: NPJ Regenerative Medicine

Article Title: Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal

doi: 10.1038/npjregenmed.2016.17

Figure Lengend Snippet: Cxcl14 knockdown enhances C2C12 differentiation. ( a ) C2C12 cells were infected with lentiviruses expressing shCxcl14 or shScramble (negative control), selected for 2 days then differentiated for 72 h, followed by staining for MHC (green) and DAPI (pseudocoloured red) ( n =3). Scale bar: 50 μm. ( b ) Cells treated as in ( a ) were differentiated and at indicated time points of differentiation (‘Hrs diff’) were lysed and subjected to western analysis ( n =3). ( c ) Densitometry was performed on blots in ( b ), and the relative levels of proteins using tubulin as reference were quantified. Paired t test was performed to compare control and shCxcl14 at each time point. * P <0.05; ** P <0.01. ( d ) C2C12 cells were treated as in ( a ), then grown in the presence or absence of 25 ng/ml recombinant Cxcl14 (rCxcl14) for 24 h after selection. Cells were then differentiated for 3 days, followed by staining for MHC and DAPI and quantification of fusion index ( n =4). Scale bar: 50 μm. Paired two-tailed t -test was performed for the data in ( d ). The data denoted by different letters ( a – c ) are significantly different from each other ( P <0.05). All error bars represent s.d. of independent replicates.

Article Snippet: U0126 and ELISA kit for detection of mouse Cxcl14 was from Thermo Scientific.

Techniques: Knockdown, Infection, Expressing, Negative Control, Staining, Western Blot, Control, Recombinant, Selection, Two Tailed Test

Journal: NPJ Regenerative Medicine

Article Title: Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal

doi: 10.1038/npjregenmed.2016.17

Figure Lengend Snippet: Cxcl14 promotes cell cycle progression. ( a ) C2C12 cells were infected with lentiviruses expressing shCxcl14 or shScramble, selected for 2 days, grown in the presence or absence of 25 ng/ml rCxcl14 for 24 h, followed by differentiation for 24 h and subsequent staining with DAPI. Stained nuclei were counted ( n =4). ( b ) Cells were treated as in ( a ) but only stimulated with 10 ng/ml rCxcl14 for the indicated amount of time. Cells were then lysed and subjected to western analysis ( n =3). ( c ) Cells were treated as in ( a ) but differentiated for 24 h, followed by BrdU incorporation for 2 h. Cells were immunofluorescently stained for BrdU and DAPI, then counted ( n =5). ( d ) Cells were treated as in ( a ), followed by TUNEL assay to detect apoptotic cells ( n =3). ( e and f ) Cells were treated as in ( a ) but grown in the presence or absence of Ara-C for 24 h, then differentiated and immunofluorescently stained for MHC (green) or DAPI (pseudocoloured red). The fusion index was calculated ( n =3). Scale bar: 50 μm. Paired two-tailed t -test was performed for all the data. The data denoted by different letters ( a – c ) are significantly different from each other ( P <0.05). All error bars represent s.d. of independent replicates.

Article Snippet: U0126 and ELISA kit for detection of mouse Cxcl14 was from Thermo Scientific.

Techniques: Infection, Expressing, Staining, Western Blot, BrdU Incorporation Assay, TUNEL Assay, Two Tailed Test

Journal: NPJ Regenerative Medicine

Article Title: Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal

doi: 10.1038/npjregenmed.2016.17

Figure Lengend Snippet: Cxcl14 knockdown accelerates muscle regeneration post-injury. ( a ) TA muscles were co-injected with BaCl 2 and shRNA viruses, and isolated on days 5, 7 and 14 after injury (AI). Upon cryosection, H&E staining was performed and regenerating myofiber cross-sectional area (CSA) was quantified ( n =6 for D5AI, n =8 for D7AI, n =7 for D14AI). ( b ) TA muscles injected as above were isolated on day 3 AI, cryosectioned and immunostained for Cxcl14 (green) along with DAPI (blue) ( n =3). Fluorescence intensity was quantified using ImageJ software. ( c – e ) TA muscle sections as described in ( b ) were immunostained for Ki-67 ( c ), MyoD ( d ) or myogenin ( e ), and the percentage of positive cells was quantified ( n =5). Scale bars: 50 μm. Paired two-tailed t -test was performed. The data denoted by different letters ( a – c ) are significantly different from each other ( P <0.05). All error bars represent s.d. of independent replicates.

Article Snippet: U0126 and ELISA kit for detection of mouse Cxcl14 was from Thermo Scientific.

Techniques: Knockdown, Muscles, Injection, shRNA, Isolation, Staining, Fluorescence, Software, Two Tailed Test

Journal: NPJ Regenerative Medicine

Article Title: Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal

doi: 10.1038/npjregenmed.2016.17

Figure Lengend Snippet: Cxcl14 enhances muscle regeneration in aging mice. ( a ) TA muscles were injured with BaCl 2 , and isolated on day 7 AI. Upon cryosection, H&E staining was performed and regenerating myofiber cross-sectional area (CSA) was quantified ( n =12 for 4 months, n =9 for 6 months, n =11 for 8 monrhs, n =12 for 12 months, n =6 for 18 months). ( b ) TA muscle sections as described in ( a ) were isolated on day 7 AI, and the RNA was isolated and quantified by qRT-PCR. Horizontal bars represent the mean of individually plotted data points ( n =15 for 4 months, n =11 for 6 months, n =12 for 8 months, n =12 for 12 months, n =6 for 18 months). ( c ) TA muscles from 2-month-old (young) and 12-month-old (aged) mice were co-injected with Cxcl14 or scramble shRNA viruses together with BaCl 2 , then processed as in ( a ) at day 7 and day 14 AI ( n =6 for each). One-way ANOVA was used to analyze the data in ( b ) and paired two-tailed t -test was performed in ( c ). The data denoted by different letters ( a – c ) are significantly different from each other ( P <0.05). All error bars represent s.d. of independent replicates. Scale bar: 50 μm.

Article Snippet: U0126 and ELISA kit for detection of mouse Cxcl14 was from Thermo Scientific.

Techniques: Muscles, Isolation, Staining, Quantitative RT-PCR, Injection, shRNA, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Plasma CXCL14 as a Candidate Biomarker for the Diagnosis of Lung Cancer

doi: 10.3389/fonc.2022.833866

Figure Lengend Snippet: Plasma CXCL14 as a diagnostic biomarker in distinguishing lung cancer patients from control subjects. (A) Comparison of the CXCL14 concentration (determined by ELISA) in plasma between control subjects (n=80) and lung cancer patients (n=286) in a retrospective cohort. P <0.0001 determined by Mann–Whitney U tests. (B) ROC analysis of the diagnostic efficiency of CXCL14 in control subjects versus lung cancer patients in a retrospective cohort (AUC=0.9464, 95% CI: 0.9209–0.9719). (C) Comparison of the CXCL14 concentration (determined by ELISA) in plasma between control subjects (n=80) and stage I lung cancer patients (n=137) in a retrospective cohort. P <0.0001 determined by Mann–Whitney U tests. (D) ROC analysis of the diagnostic efficiency of CXCL14 in control subjects versus stage I lung cancer patients in a retrospective cohort (AUC=0.9353, 95% CI: 0.9034–0.9672). Scatter diagrams present the median values with interquartile ranges.

Article Snippet: The CXCL14 level in plasma and urine was assayed using commercially available sandwich enzyme-linked immunosorbent assay kits, Human CXCL14/BRAK DuoSet ELISA and DuoSet ELISA Ancillary Reagent Kit 2(Lot DY866&DY008, R&D Systems).

Techniques: Clinical Proteomics, Diagnostic Assay, Biomarker Discovery, Control, Comparison, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Plasma CXCL14 as a Candidate Biomarker for the Diagnosis of Lung Cancer

doi: 10.3389/fonc.2022.833866

Figure Lengend Snippet: Urinary CXCL14 as a diagnostic biomarker in distinguishing lung cancer patients from control subjects. (A) Comparison of the CXCL14 concentration (determined by ELISA) in urine between control subjects (n=122) and lung cancer patients (n=284) in a retrospective cohort. P <0.0001 determined by Mann–Whitney U tests. (B) ROC analysis of the diagnostic efficiency of urinary CXCL14 in a retrospective cohort of control subjects versus lung cancer patients (AUC=0.6476, 95% CI: 0. 0.5934–0.7091, P <0.0001). Scatter diagrams present the median values with interquartile ranges. (C) Comparison of the CXCL14 concentration (determined by ELISA) in urine between control subjects (n=122) and stage I lung cancer patients (n=158) in a retrospective cohort. P <0.0001 determined by Mann–Whitney U tests. (D) ROC analysis of the diagnostic efficiency of CXCL14 in control subjects versus stage I lung cancer patients in a retrospective cohort (AUC=0.647, 95% CI: 0.5829–0.7111). Scatter diagrams present the median values with interquartile ranges.

Article Snippet: The CXCL14 level in plasma and urine was assayed using commercially available sandwich enzyme-linked immunosorbent assay kits, Human CXCL14/BRAK DuoSet ELISA and DuoSet ELISA Ancillary Reagent Kit 2(Lot DY866&DY008, R&D Systems).

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