Journal: Oncotarget

Article Title: Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

doi: 10.18632/oncotarget.13446

Figure Lengend Snippet: ( A ) Graph demonstrating the relative RARRES2 (Chemerin gene) mRNA expression in senescent (SEN) vs. young (YNG) fibroblast of different strains (FF95, FFRa and FFPia) as defined by qRT-PCR. Data are normalized to the expression level of RARRES2 in keratinocytes, confirming that the senescent fibroblasts display the highest, and the cSCC cell lines (SCL-1, SCC12-B2, SCC-13) display the lowest RARRES2 transcripts, respectively. Date are shown as mean ± S.D for one of three independent experiments of biological replicates ( n = 3); * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA. ( B ) Chemerin secretion was analyzed in the above mentioned cells (normalized to 5 × 10 6 cells/ml) using ELISA. Data are shown as mean ± S.E.M for three independent experiments; * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA. (Note that due to low standard deviations of some measurements, error bars are not visible for all data points.) ( C ) Representative photomicrographs of paraffin-embedded human skin sections co-immunostained with anti-FSP-1 antibody in green and anti-Chemerin antibody in red, depicting higher abundance of Chemerin in skin dermal fibroblasts of aged (70-year old), compared to young (23-year old) donors. Nuclei were DAPI-counterstained (blue). Appropriate isotype controls were used to determine the background. Scale bars = 50 μm at ×400 magnification; Dashed lines delineate epidermis (E) from dermis (D). Orange arrows point to the Chemerin-positive fibroblasts. Orange boxes depict the magnified area. ( D ) Graph representing the quantification of Chemerin-positive fibroblasts (shown by FSP-1 marker) in the skin dermis of old healthy individuals (76 ± 10 year, n = 15 donors) and young (21 ± 8 year, n = 13 donors) calculated from minimum 5 technical replicates. *** p < 0.001 by two-tailed student t -test.

Article Snippet: Recombinant human (rh) Chemerin (#2324-CM/CF) and rh RANTES/CCL5 (#278-RN/CF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Marker, Two Tailed Test

Journal: Oncotarget

Article Title: Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

doi: 10.18632/oncotarget.13446

Figure Lengend Snippet: Graphs demonstrating the bell-shape curves of chemotactic response to the increased concentrations of Chemerin in ( A ) SCL-1, ( B ) SCC-12B2, ( C ) SCC-13 and ( D ) A431 cells using the Transwell ® chamber migration assay. Date are shown as mean ± S.D for one of three independent experiments with n = 4 replicate wells; n.s. = non-significant, * p < 0.05, ** p < 0.01 and *** p < 0.001 in comparison to random migration control with no Chemerin treatment calculated by Bonferroni post hoc test after ANOVA. The role of Chemerin in mediating cSCC cell migration was confirmed by silencing the Chemerin gene (RARRES2) in senescent fibroblasts and assessing their ability to induce SCL-1 cell migration ( E ) Transcript level of RARRES2 was quantified by qRT-PCR in senescent fibroblasts 42 h post treatment with siRNAs, confirming the successful gene silencing compared to scrambled control. Data are shown as mean ± S.E.M from three independent experiments. *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA. ( F ) Chemerin protein level (normalized to 5 × 10 6 cells/ml) was measured 48 hours after treatment with siRNAs or scrambled control. Data are shown as mean ± S.E.M from four independent experiments; * p < 0.05 calculated by Bonferroni post hoc test after ANOVA. ( G ) Conditioned media derived from Chemerin-silenced fibroblasts (siRNA 1, 2, 5 and 6) were used to induce the migration of SCL-1 cells using the Transwell ® chamber migration assay. Conditioned media derived from mock-treated fibroblasts (Scrambled) and senescent fibroblasts with no treatment (control) were used as controls. Shown is one representative of four independent experiments, each with triplicate samples. * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA.

Article Snippet: Recombinant human (rh) Chemerin (#2324-CM/CF) and rh RANTES/CCL5 (#278-RN/CF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Comparison, Control, Quantitative RT-PCR, Derivative Assay

Journal: Oncotarget

Article Title: Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

doi: 10.18632/oncotarget.13446

Figure Lengend Snippet: ( A ) Heat map representing the gene expression profile of Chemerin receptors CCRL2, GPR1 and CMKLR1. Relative gene expression was calculated from all three independent experiments normalized to the level of Actin housekeeping gene expression, and relative to the mean expression in normal keratinocytes. Genes were rank-ordered by Pearson correlation and depicted by a pseudo color scale based on qRT-PCR delta-CT values with green and red demonstrating low and high mRNA abundance, respectively. CMKLR1 had very low to no mRNA transcripts; CCRL2 was highly upregulated in cSCC cells; and GPR1 was either downregulated or not changed in cSCC cells compared to keratinocytes. ( B – C ) Graphs depict the relative fold changes of CCRL2 and GPR1 mRNA expression in cSCC lines compared with normal keratinocytes as defined by qRT-PCR. Shown is one representative of three independent experiments, each with triplicate samples. * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA. (Note that due to low standard deviations of some measurements, error bars are not visible for all data points.) ( D – E ) Cell surface expressions of CCRL2, GPR1 and CMKLR1 were assessed by flow cytometry analysis in SCL-1 cells and normal keratinocytes. Displayed are the representative fluorescence histograms depicting the relative fluorescence intensity of cells stained with (D) APC-conjugated anti-CCRL2 and (E) A488-conjugated anti-GPR1 antibodies (black histograms) compared with isotype control antibodies conjugated with corresponding fluorochromes (gray histograms). The expression of CMKLR1 was not detectable (not shown). Bar charts represent one of three independent experiments demonstrating the mean percentage ± S.D. for (d) CCRL2-positive and (E) GPR1-positive SCL-1 cells vs. keratinocytes. * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by two-tailed student t -test ( n = 8 biological replicates). ( F ) Representative Western bot analysis of cSCC and keratinocyte cell lysates, confirming the elevated protein level of CCRL2 in cSCC cells compared with normal keratinocytes. ( G ) Representative photomicrographs of skin biopsies derived from patients suffering from invasive cSCC vs. normal healthy controls stained for Hematoxylin and Eosin (H & E), CCRL2 in red and cytokeratin in green. Nuclear was stained with DAPI (blue). Arrows show the CCRL2-positive cells. Appropriate isotype controls were used to determine the background. H&E images were taken at ×200 magnification and immunofluorescence at ×400 magnification; Scale bars = 50 μm. Orange boxes depict the magnified area. ( H ) Graph representing the quantification of CCRL2-positive epidermal-derived cells in the skin specimens of patients suffering from cSCC ( n = 8) vs. normal healthy individuals ( n = 6). ** p < 0.01 by two-tailed student t -test.

Article Snippet: Recombinant human (rh) Chemerin (#2324-CM/CF) and rh RANTES/CCL5 (#278-RN/CF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Staining, Control, Two Tailed Test, Western Blot, Derivative Assay, Immunofluorescence

Journal: Oncotarget

Article Title: Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

doi: 10.18632/oncotarget.13446

Figure Lengend Snippet: ( A ) In order to define the signaling pathway activated by Chemerin, we used a G-protein couple receptor (GPCR) Cignal Finder ® Reporter Array consisting of inducible transcription factor response constructs encoding the firefly luciferase reporter gene. SCL-1 cells were transfected with each reporter constructs and further treated with DMEM containing 40 nM rh Chemerin or no Chemerin (control) for 1 hour. The dual-luciferase assay was developed and the results were expressed as the percentage of relative luminescence signal according to the manufacturer's instructions. Results highlighted that Chemerin activates JNK and ERK1/2 MAPK signaling pathway. Shown is a representative of three independent experiments presented as Mean ± S.D ( n =3). * p < 0.05 calculated by unpaired student t -test. (Note that due to low standard deviations of some measurements, error bars are not visible for all data points.) ( B ) To confirm the Cignal Finder ® Reporter Array data, SCL-1 cells were cultured in the presence and absence of 40 nM rh Chemerin for 30 and 60 minutes and protein lysates were analyzed by Western blot for phosphorylated and total amounts of MAPK key proteins. ( C ) We investigated whether inhibition of MAPK pathway dampens the migration of SCL-1 cells stimulated by Chemerin-rich conditioned media of senescent fibroblasts. The graph represents the concentration-dependent inhibition of SCL-1 cell migration in response to senescent fibroblast CM by SP600125 (JNK inhibitor), FR180204 (ERK1/2 inhibitor) and BIRB796 (P38 inhibitor). DMSO served as a vehicle control. Data are presented as Mean ± S.E.M. for 3 independent experiments; * p < 0.05, ** p < 0.01 and *** p < 0.001 in comparison to DMSO control calculated by Bonferroni post hoc test after ANOVA; HPF= ×100 magnification ( D ) Bar graph showing the effect of MAPK inhibitors (10 μM) on Chemerin-mediated SCL-1 cell migration. Data are presented as mean ± S.E.M. for 3 independent experiments; *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA. HPF= ×100 magnification.

Article Snippet: Recombinant human (rh) Chemerin (#2324-CM/CF) and rh RANTES/CCL5 (#278-RN/CF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Construct, Luciferase, Transfection, Control, Cell Culture, Western Blot, Inhibition, Migration, Concentration Assay, Comparison

Journal: Oncotarget

Article Title: Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

doi: 10.18632/oncotarget.13446

Figure Lengend Snippet: In order to study the role of Chemerin receptors in mediating cSCC cell migration, SCL-1 cells were transduced with lentivirus encoding shRNAs against GPR1 and CCRL2. The GPR1 shRNA-expressing lentiviral vector and the CCRL2 shRNA-expressing lentiviral vector contained mCherry and eGFP proteins, respectively, which enabled us to isolate positively-transduced cells using fluorescence-activated cell sorting (FACS). ( A – B ) Representative FACS dot plots showing the final gating strategies to sort mCherry- and eGFP-expressing cells. ( C – D ) The efficiency of CCRL2 and GPR1 knockdown in SCL-1 cells was validated by qRT-PCR. Data are presented as bar charts, representing the mean expression fold changes relative to scrambled controls ± S.D. * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by ANOVA ( n = 3). ( E ) Graph indicating that CCRL2 and GPR1 silencing suppresses Chemerin-mediated SCL-1 cell migration. The relative migration ( Chemerin / Untreated ) was calculated in each group as the ratio of cells migrated towards the Chemerin gradient (40 nM) to the cells migrated randomly in the negative control (serum-free DMEM). Data are presented as Mean ± S.E.M. for 3 independent experiments; * p < 0.05, ** p < 0.01 and *** p < 0.001 calculated by Bonferroni post hoc test after ANOVA.

Article Snippet: Recombinant human (rh) Chemerin (#2324-CM/CF) and rh RANTES/CCL5 (#278-RN/CF) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Transduction, shRNA, Expressing, Plasmid Preparation, Fluorescence, FACS, Knockdown, Quantitative RT-PCR, Negative Control