Journal: PloS one

Article Title: Interaction between CXCR4 and CCL20 pathways regulates tumor growth.

doi: 10.1371/journal.pone.0005125

Figure Lengend Snippet: Figure 1. CXCR4 up-regulates CCL20 production by prostate cancer cell line PC3. (A–C) Wild-type PC3 cells, CXCR4-transduced PC3 cell line and single cell clone with stable overexpression of CXCR4 (PC3-CXCR4.5) were stained for the control (IgG2a-PE, purple) and CXCR4 antibodies (IgG2a-12G5, green) and analyzed using FACS. PC3 and PC3-CXCR4.5 cells were stimulated with CXCL12 at concentrations of 50, 250 and 1,000 ng/ml for 48 hours, harvested and viable cells were counted using PI staining and FACS analysis. PC3 and PC3-CXCR4.5 cells were (56106/mice) were injected subcutaneously into SCID/beige mice. 60 days following the injection, animals were sacrificed, tumor size (cm2) and tumor weight (g) were measured. Data is presented as mean6SE from 5 mice. (D) PC3 and PC3-CXCR4.5 cells were stimulated with CXCL12 at concentrations 5 and 500 ng/ ml for 24 hours, total RNA was extracted, reverse-transcribed and subjected to quantitative PCR for CCL20. PCR analysis was carried out in triplicates. (E) PC3 and PC3-CXCR4.5 cells were stimulated with various concentrations of CXCL12 (5, 50, 25, 500, and 1,000 ng/ml) for 48 hours and CCL20 secretion was assessed by ELISA. The results represent the average of triplicates6SD (** P,0.05). (F) PC3-CXCR4.5 cells were incubated with CXCL12 at concentration of 500 ng/ml. At the indicated time points CCL20 was assessed in extra-cellular (culture medium) and intra-cellular (whole cell lysate) fractions using ELISA method. (G) In order to inhibit CXCR4 signaling, PC3 and PC3-CXCR4.5 cells were cultured with anti-CXCL12 antibodies or pertussis toxin alone or in combination with CXCL12 during 48 hours, and CCL20 secretion was assessed by ELISA. CCL20 secretion in PC3 and PC3- CXCR4.5 cells was also inhibited using JAK-2 inhibitor AG-490 at 1 mm/ml. and the MEK inhibitor- PD98059 (20 mM). The results represent the average of triplicates6SD (** P,0.05). doi:10.1371/journal.pone.0005125.g001

Article Snippet: For the neutralizing experiments, mice were treated with subcutaneous injections of monoclonal anti-human CCL20 antibody (MAB360, R&D Systems, Minneapolis, MN) or control IgG1 antibody.

Techniques: Over Expression, Staining, Control, Injection, Reverse Transcription, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Cell Culture

Journal: PloS one

Article Title: Interaction between CXCR4 and CCL20 pathways regulates tumor growth.

doi: 10.1371/journal.pone.0005125

Figure Lengend Snippet: Figure 2. Regulation of CCL20 expression and function. (A–B) CXCR4 expression levels of PC3-CXCR4.5 cells 48 hours following the transfection with control non-specific siRNA and specific anti-CXCR4 siRNA. The cells were stained for the control and CXCR4 antibodies and evaluated by FACS. Semi-quantitative RT-PCR analysis of CXCR4 and CCL20 mRNA of the siRNA-transfected PC3-CXCR4.5 cells 48 hours following the transfection. b-actin confirmed comparable loading of RT-PCR products in each lane. Control and CXCR4 siRNA-transfected PC3-CXCR4.5 cells at 48 hours post-transfection were incubated with CXCL12 500 ng/ml for an additional 48 hours. CCL20 secretion was assessed by ELISA. The results represent the average of triplicates6SD (** P,0.05). (C) CCR6 expression in prostate cancer cell lines PC3, LaNCAP, 22Rv1 and DU145 was evaluated by FACS and PCR whereas CCL20 expresion was evaluated by PCR and ELISA assays. Purple line represents mouse IgG control antibody, green line represents staining with CCR6 monoclonal antibody. (D) PC3 and PC3-CXCR4 cells were incubated with various concentration of CCL20 for 6 days. Following 3 days of incubation, the medium with or without CCL20 was renewed. On day 6, the cells were harvested and viable cells were counted using PI staining and FACS analysis. In addition, in order to determine proliferation of PC3 cells, the cells were labeled with BrdU (10 mM) during the last 16 hours of incubation and processed for BrdU detection using specific anti-BrdU FITC-conjugated antibody and FACS analysis. Data is presented as mean6SD from triplicates (** P,0.05). Data is representative of two separate experiments. (E–F) PC3 and PC3-CXCR4.5 cells either untreated or treated with various concentrations of CCL20 were placed on collagen I- or fibronectin-coated plates (10 mg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.05). In addition, PC3 cells (E) that demonstrated increased adhesion to collagen I and fibronectin in response to stimulation with CCL20, were co-incubated with CCL20 and PTX (100 ng/ml) and were allowed to adhere to collagen I- and fibronectin-coated plates. doi:10.1371/journal.pone.0005125.g002

Article Snippet: For the neutralizing experiments, mice were treated with subcutaneous injections of monoclonal anti-human CCL20 antibody (MAB360, R&D Systems, Minneapolis, MN) or control IgG1 antibody.

Techniques: Expressing, Transfection, Control, Staining, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Labeling

Journal: PloS one

Article Title: Interaction between CXCR4 and CCL20 pathways regulates tumor growth.

doi: 10.1371/journal.pone.0005125

Figure Lengend Snippet: Figure 3. Regulation of CCL20 expression and function in various tumor cells. (A) CCL20 mRNA and protein expression in CCL20- transfected PC3 single-cell clones tested by semi-quantitative RT-PCR and ELISA. (B) PC3-CCL20 clones were seeded at 26104 cells/1 ml per well into a 24-well plate and incubated for 6 days. On day 6, the cells were harvested and viable cells were counted using PI staining and FACS analysis. Data is presented as mean6SD from triplicates (** P,0.05). (C) PC3-CCL20 single-cell clones were grown to confluence, harvested, resuspended in their conditioned culture medium and allowed to adhere to collagen I- and fibronectin-coated plates for 30 minutes. Non-adherent cells were washed, and adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.05). (D) Leukemic cell lines NB4 and HL60, primary human leukemic blasts, HT-29 cells, and normal human keratinocytes were incubated with various concentrations of CXCL12 for 48 hours. CCL20 secretion to culture medium was assessed using ELISA method. (E) CCR6 mRNA expression in leukemic cell lines NB4 and HL60 and colon cancer HT-29 cells assessed by semi-quantitative RT-PCR. b-actin confirmed comparable loading of RT- PCR products in each lane. (F) HL60 and HT-29 cells either untreated or treated with various concentrations of CCL20 were placed on collagen I coated plates (10 mg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.05). Data is representative of three separate experiments. doi:10.1371/journal.pone.0005125.g003

Article Snippet: For the neutralizing experiments, mice were treated with subcutaneous injections of monoclonal anti-human CCL20 antibody (MAB360, R&D Systems, Minneapolis, MN) or control IgG1 antibody.

Techniques: Expressing, Transfection, Clone Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: PloS one

Article Title: Interaction between CXCR4 and CCL20 pathways regulates tumor growth.

doi: 10.1371/journal.pone.0005125

Figure Lengend Snippet: Figure 4. CCL20 regulates CXCR4 dependent and independent growth of tumor cells. (A) Effect of CCL20 stable expression on prostate tumor growth. PC3-CCL20.30, PC3-CCL20.10 and PC3-mock transfected cells (56106/mouse) were injected subcutaneously into SCID/beige mice. 48 days or 75 days following the injection, animals were sacrificed, and xenograft tumors generated by PC3-mock and PC3-CCL20.30 injected cells were purified . Tumor size (cm2) was measured twice a week using caliper. Results are representative of three independent experiments with five mice in each group. Data is presented as mean6SE from five mice. (B) H&E staining of paraffin-embedded tumor tissue sections derived from PC3-mock and PC3-CCL20.30 tumors on day 48. Black arrows signify non-invasive borders of PC3-mock tumor (B,a), small blood vessel in PC3-mock tumor (B,b), aberrant blood vessels in PC3-CCL20.30 tumor (B, c,d), original magnification of 6200 is shown. (C) Vessel functionality (DSo2) was measured by fMRI. Functionality of the vasculature was tested during inhalation of air-CO2 and carbogen (95% oxygen+5% CO2) in mice implanted with PC3-mock cells or with PC3-CCL20.30 cells. DSo2 values from PC3-mock cells and PC3-CCL20.30 are shown. The mean6SD values of DSo2 were calculated from whole tumor, and normalized to contra-lateral muscle, pooling data from 9 mice from the PC3-CCL20.30 group and 5 mice from the PC3-mock group (four slices/mouse; p,0.001). (D) Adhesion of PC3-CCL20.30 cells to collagen I. PC3-CCL20.30 cells either umstimulated or stimulated with 50 ng/ml of CCL20 with or without co-incubation with neutralizing anti-CCL20 antibodies (10 mg/ml) were placed on collagen I-coated plates (10 mg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 ml FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean6SD from triplicates (** P,0.016). (E) PC3-CCL20.30 cells (56106/mouse) were injected subcutaneously into SCID/ beige mice. Twenty-four hours after the cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 mg of antibody per injection, three times a week, during four weeks. Tumor size (cm2) was measured once a week using caliper. Results are representative of two independent experiments with ten mice in each group. Data is presented as mean6SE from ten mice. (F) On day 64 following the cell injection, the experiment was terminated, animals were sacrificed; subcutaneous tumors were removed, measured and weighted. Data is presented as mean6SE from ten mice in each group (** P,0.0002). (G) PC3-CXCR4.5 cells (56106/mouse) were injected subcutaneously into SCID/beige mice. Twenty-four hours after the cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 mg of antibody per injection, three times a week, during four weeks. Tumor size (cm2) was measured once a week using caliper. On day 55 following cell injection, the experiment was terminated; animals were sacrificed, subcutaneous tumors were removed, measured and weighted. Data is presented as mean6SE from ten mice in each group (** P,0.0027). (H) HT-29 cells (26106/mouse) were injected subcutaneously into nude mice. Twenty-four hours after cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 mg of antibody per injection, five times a week, during two weeks. On day 17 following cell injection, the experiment was terminated, animals were sacrificed; subcutaneous tumors were removed, measured and weighted. Data is presented as mean6SE from ten mice in each group (** P,0.0002). doi:10.1371/journal.pone.0005125.g004

Article Snippet: For the neutralizing experiments, mice were treated with subcutaneous injections of monoclonal anti-human CCL20 antibody (MAB360, R&D Systems, Minneapolis, MN) or control IgG1 antibody.

Techniques: Expressing, Transfection, Injection, Generated, Purification, Staining, Derivative Assay, Incubation, Control

Journal: PloS one

Article Title: Interaction between CXCR4 and CCL20 pathways regulates tumor growth.

doi: 10.1371/journal.pone.0005125

Figure Lengend Snippet: Figure 5. CCL20, CCR6 and CXCR4 expression in prostate cancer cell lines, in primary prostate tumor tissue and in normal prostate tissue. (A) Expression of CCL20 and CCR6 in human prostate cancer tissue and in human normal prostate tissue. Immunohistostaining of prostate cancer and normal specimens using the polyclonal antibody for CCL20 and the monoclonal antibody 140706 for CCR6. Original magnification of 6400 is shown. CCL20 and CCR6 expression was observed in endothelial and fibromuscular cells of prostate samples (signed with black arrows). A commercially available array including 52 samples (CA2) from patients with prostate cancer was stained for CCL20 (A9) and CCR6 (A0). Expression was scored on two levels: low or negative expression, and high expression. Statistical analysis of the immunohistochemical staining was performed using two-tailed Mann-Whitney test. (B) Comnmon expression pattern of CCL20 and CXCR4 in human prostate cancer. Immunohistostaining of prostate cancer specimens using the polyclonal antibody for CCL20 and the monoclonal antibody 12G5 for CXCR4. Original magnification of 6400 is shown. CCL20 and CXCR4 expression was observed in endothelial and fibromuscular cells of prostate samples. (B9) A commercially available array including 48 samples (CA3) from patients with prostate cancer was stained for CCL20 and CXCR4. Expression was scored on three levels: low, intermediate, and high expression. Samples that demonstrated the same scoring in CCL20 and CXCR4 expression levels were classified as having similar expression pattern. Samples where scores in CCL20 and CXCR4 expression differed in one level or more were classified as different expression patterns. doi:10.1371/journal.pone.0005125.g005

Article Snippet: For the neutralizing experiments, mice were treated with subcutaneous injections of monoclonal anti-human CCL20 antibody (MAB360, R&D Systems, Minneapolis, MN) or control IgG1 antibody.

Techniques: Expressing, Staining, Immunohistochemical staining, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Biological Chemistry

Article Title: The Haemophilus influenzae Hia Autotransporter Contains an Unusually Short Trimeric Translocator Domain

doi: 10.1074/jbc.m311496200

Figure Lengend Snippet: FIG. 2. Immunoblot and functional analysis of Hia deletion mutants expressed in E. coli BL21(DE3). A, outer membrane pro- teins detected by Western blot with a guinea pig antiserum directed against Hia residues 50–252. B, flow cytometry analysis of Hia on the bacterial surface performed with a guinea pig antiserum against Hia residues 221–658. Strain BL21(DE3)/pT7-7 (vector) was used as the negative control, and the corresponding histogram was superimposed (solid line) on results with E. coli expressing wild-type Hia (pHMW8-7) or the Hia deletion derivatives (filled histograms). C, adherence to Chang epithelial cells by E. coli BL21(DE3) expressing the indicated constructs. Bars represent the means S.E.

Article Snippet: Quantitative Adherence Assays—Adherence assays were performed with Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva, ATCC CCL20.2) or with A549 respiratory epithelial cells (ATCC CCL185) as described previously (23).

Techniques: Western Blot, Functional Assay, Membrane, Flow Cytometry, Plasmid Preparation, Negative Control, Expressing, Construct

Journal: Journal of Biological Chemistry

Article Title: The Haemophilus influenzae Hia Autotransporter Contains an Unusually Short Trimeric Translocator Domain

doi: 10.1074/jbc.m311496200

Figure Lengend Snippet: FIG. 5. Identification of Yersinia sp. YadA and N. meningitidis NhhA as autotransporters. Adherence to A549 epithelial cells by H. influenzae DB117 expressing the indicated proteins. Bars represent the means S.E.

Article Snippet: Quantitative Adherence Assays—Adherence assays were performed with Chang epithelial cells (Wong-Kilbourne derivative, clone 1-5c-4, human conjunctiva, ATCC CCL20.2) or with A549 respiratory epithelial cells (ATCC CCL185) as described previously (23).

Techniques: Expressing

Journal: RNA

Article Title: MicroRNA-mediated regulation of extracellular matrix formation modulates somatic cell reprogramming

doi: 10.1261/rna.043745.113

Figure Lengend Snippet: Wisp1 is a key regulator of extracellular matrix genes. ( A ) Wisp1 regulates expression of several ECM genes. Expression of Tgfbi, Igfbp5, Dkk2, Nov, and Ccl20 were dramatically changed upon Wisp1 knockdown. Uninfected and 4F-infected MEFs were transfected with siWisp1 for 2 d and total RNAs were harvested for RT-qPCR analysis of different ECM genes. Error bars represent two independent experiments with duplicate wells. ( B ) Knockdown of Nov, Dkk2, and Tgfbi significantly enhances iPSC generation. MEFs were transduced with 4F at Day 0 and transfected with siRNAs at Day 5 post-infection. GFP + colonies were quantified at around Days 11–13. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. ( C ) Overexpression of Wisp1-regulated ECM genes compromises reprogramming. The indicated ECM genes were cloned into pMX retroviral vectors. MEFs were transduced with 4F plus the indicated ECM genes and GFP + colonies were quantified at around Days 11–13. Data were normalized to pMX-RFP-transduced cells. Error bars represent three independent experiments with triplicate wells. (**) P < 0.01. ( D ) Addition of recombinant ECM proteins compromises reprogramming. Purified recombinant TGFBI, DKK2, NOV, and CCL20 were added at a final concentration of 100 ng/mL to cultures of 4F-MEFs undergoing reprogramming. GFP + colonies were quantified at Days 11–13. Error bars represent two independent experiments with triplicate wells. (*) P < 0.05.

Article Snippet: Recombinant proteins were obtained from commercial sources as follows: mouse Dkk2 (R&D systems, 2435DK/CF), human NOV/CCN3 (R&D systems, 1640NV), human TGFBI (Prospec, #PRO-568), CCL20 (R&D systems, 760-M3).

Techniques: Expressing, Knockdown, Infection, Transfection, Quantitative RT-PCR, Transduction, Over Expression, Clone Assay, Retroviral, Recombinant, Purification, Concentration Assay

Journal: Cell Death & Disease

Article Title: Gasdermin E promotes translocation of p65 and c-jun into nucleus in keratinocytes for progression of psoriatic skin inflammation

doi: 10.1038/s41419-024-06545-5

Figure Lengend Snippet: A Neutrophil infiltration was evaluated by detecting Ly6G and MPO through immunohistochemistry study. Statistical analysis of mean staining intensity was shown. B Quantitative real-time PCR analysis was performed to determine the mRNA levels of Il17a, Il23a, Tnfa, Il1b, Ccl20, Cxcl1, Cxcl2, S100a8, and S100a9 . Statistical analysis was shown. C The percentage of CD4+T cells (CD3+CD4+), Th1 cells (CD3+CD4+IFN-γ+), and Th17 cells (CD3+CD4+IL-17A+) in spleen was determined by flow cytometry. Statistical analysis of specific cell percentages was shown. Scale bar represents 100 μm in A . ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ELISA kits of S100A8/A9 (KE00177), CCL20 (KE00149), and IL-8 (KE00006) were purchased from Proteintech (Wuhan, China).

Techniques: Immunohistochemistry, Staining, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Gasdermin E promotes translocation of p65 and c-jun into nucleus in keratinocytes for progression of psoriatic skin inflammation

doi: 10.1038/s41419-024-06545-5

Figure Lengend Snippet: A Proteins of GSDME FL and GSDME NT were detected by western blotting in HaCaT cells at 12, 24, and 48 h after treatment with or without M5. Statistical analysis of interested protein levels was shown. B HaCaT cells were transfected with lentivirus vector containing either NC shRNA or GSDME shRNA, establishing GSDME knockdown cells (sh GSDME) and control cells (sh NC). Knockdown efficiency was evaluated using western blotting assay. Statistical analysis of GSDME was shown. C , D Immunofluorescence assay was used to detect Ki-67 expression or Edu-positive cells. Statistical analysis of mean immunofluorescence intensity was shown. E Quantitative real-time PCR analysis was performed to determine mRNA levels of Il1b, Ccl20, Cxcl1, Cxcl8, S100a8 , and S100a9 . Statistical analysis was shown. F Proteins of GSDME FL, S100A8, and S100A9 in cell lysate were detected by western blotting assay. Statistical analysis of interested protein levels was shown. G Secretion levels of S100A8/A9, CCL20, and CXCL8 were determined by ELISA assay. Statistical analysis was shown. H S100A8 and S100A9 were detected by immunofluorescence assay in mice skin sections, respectively. Statistical analysis was shown. I Proteins of p-p65, p65, p-c-jun, and c-jun in cell lysate were detected by western blotting assay. Statistical analysis of interested protein levels was shown. J Proteins of p65, c-jun, and GSDME-FL were detected by western blotting assay in lysate of cell plasma or nucleus, respectively. Statistical analysis of interested protein levels was shown. GAPDH and HDAC1 served as a loading control of cell plasma protein and nucleus proteins, respectively. K , L Immunofluorescence assay was used to detect translocation of p65 and c-jun into nucleus. M Co-immunoprecipitation assay was used to detect the interaction between GSDME-FL and p65 or c-jun. Scale bar represents 100 μm in C , D , H , K and L . ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: ELISA kits of S100A8/A9 (KE00177), CCL20 (KE00149), and IL-8 (KE00006) were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, shRNA, Knockdown, Control, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Translocation Assay, Co-Immunoprecipitation Assay

Journal: Cell Death & Disease

Article Title: Gasdermin E promotes translocation of p65 and c-jun into nucleus in keratinocytes for progression of psoriatic skin inflammation

doi: 10.1038/s41419-024-06545-5

Figure Lengend Snippet: Primer sequences in this study.

Article Snippet: ELISA kits of S100A8/A9 (KE00177), CCL20 (KE00149), and IL-8 (KE00006) were purchased from Proteintech (Wuhan, China).

Techniques:

Journal: Cells

Article Title: Protective Effects of Keratinocyte-Derived GCSF and CCL20 on UVB-Induced Melanocyte Damage

doi: 10.3390/cells13191661

Figure Lengend Snippet: The protective effects of paracrine factors on UVB-induced apoptosis, ROS formation, and melanogenesis in MC. A heat map representing the color-coded expression levels of the paracrine protective effects of KC and 14 recombinant paracrine factors on UVB-induced apoptosis ( A ), ROS production ( B ), and melanin content ( C ) in MC cells was created. The blue color represented the inhibitory action of paracrine protective effects, while the red color represented the activating action. We examined the effects of UVB (125 mJ/cm 2 ) on GCSF and CCL20 levels in KC cells. At 12 h post-irradiation, CM from KC at three cell concentrations (7 × 10 3 , 21 × 10 3 , 63 × 10 3 cells/cm 2 ) were collected, and the concentrations of GCSF ( D ) and CCL20 ( E ) were measured. The data are expressed as mean ± SD. The statistical significance of differences between UVB-irradiated KC at different cell concentrations (7 × 10 3 , 21 × 10 3 , 63 × 10 3 cells/cm 2 ) was evaluated by one-way ANOVA followed by Dunnett’s test (* p < 0.05; ** p < 0.01; *** p < 0.001 versus unirradiated control KC).

Article Snippet: GCSF and CCL20 levels in culture supernatants were determined using competitive enzyme immunoassay kits (DCS50 and DM3A00, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Recombinant, Irradiation, Control

Journal: Cells

Article Title: Protective Effects of Keratinocyte-Derived GCSF and CCL20 on UVB-Induced Melanocyte Damage

doi: 10.3390/cells13191661

Figure Lengend Snippet: The protective effects of the paracrine factors on UVB-induced transcriptional activation of MITF in MC. We examined the effects of UVB (125 mJ/cm 2 ) on tyrosinase ( A ) and TRP1 ( B ) mRNA expression in MC pretreated with CM from KC, irradiated with UVB (125 mJ/cm 2 ), GCSF, CCL20 (9 nM), and α-MSH (200 nM). MC were harvested at 1 h after UVB irradiation for the determination of ROS formation. Data are expressed as mean ± SD. The statistical significance of differences was evaluated by one-way ANOVA followed by Dunnett’s test. ** p < 0.01; *** p < 0.001 versus unirradiated cells. The statistical significance of differences between UVB-irradiated MC and UVB-irradiated MC+KC-CM, as well as GCSF and CCL20, was evaluated by one-way ANOVA followed by Dunnett’s test (# p < 0.05; ## p < 0.01; ### p < 0.001 versus UVB-irradiated MC).

Article Snippet: GCSF and CCL20 levels in culture supernatants were determined using competitive enzyme immunoassay kits (DCS50 and DM3A00, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Irradiation

Journal: Cells

Article Title: Protective Effects of Keratinocyte-Derived GCSF and CCL20 on UVB-Induced Melanocyte Damage

doi: 10.3390/cells13191661

Figure Lengend Snippet: Epidermal thickness and the expression of GCSF, CCL20, and tyrosinase protein in UVB-treated mouse skin. Epidermal thickness ( A ), immunofluorescence of GCSF ( A ), CCL20 ( C ), tyrosinase ( A , C ), and pan-cytokeratin, a marker that identifies keratinocytes in all layers of the epidermis ( A , C ), were assessed 12 h following the final UVB exposure. Red arrows indicate the epidermal layer, while white arrows mark the dermal-epidermal junction, delineating the boundary between the epidermis and dermis. The summary graph shows the statistical analysis of epidermal thickness ( B ), and the relative protein levels of GCSF ( D ), and CCL20 ( E ), to pan-cytokeratin and tyrosinase ( F ). The data were quantified using ImageJ and GraphPad Prism software (v9) and are expressed as mean ± SD, with 1 N (dot) representing one area from four areas in a mouse. A mouse provides three N. The statistical significance of differences was evaluated by one-way ANOVA followed by Dunnett’s test. *** p < 0.001 versus non-irradiated group.

Article Snippet: GCSF and CCL20 levels in culture supernatants were determined using competitive enzyme immunoassay kits (DCS50 and DM3A00, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Marker, Software, Irradiation