Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 6 An increase in levels of acetyl-CoA and H3K27ac promotes CXCL5 secretion in CAFs. The effects of acetate treatment on acetyl-CoA levels in LX-2 cells incubated with ExoHCT116 (A) and ExoSW480 (B). Western blot showed the effects of acetate treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 (C) and ExoSW480 (D). E The effects of Trichostatin A (TSA) treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoHCT116 KD and their relative control. F The effects of C646 treatment on CXCL5 and H3K27ac levels in LX-2 cells incubated with ExoSW480 OE and their relative control. G, H Chromatin immunoprecipitation (ChIP) assays using IgG as a control were performed with antibody against H3K23ac. Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl or in LX-2 cells incubated with ExoHCT116 KD and ExoHCT116 KD Ctrl and treated with TSA (G). Examination of H3K27 acetylation status in CXCL5 gene promoter region in LX-2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl or in LX- 2 cells incubated with ExoSW480 OE and ExoSW480 OE Ctrl and treated with C646 (H). Each experiment was performed in triplicate. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Incubation, Western Blot, Control, Chromatin Immunoprecipitation

Journal: Cell death & disease

Article Title: Cancer-derived exosomal HSPC111 promotes colorectal cancer liver metastasis by reprogramming lipid metabolism in cancer-associated fibroblasts.

doi: 10.1038/s41419-022-04506-4

Figure Lengend Snippet: Fig. 8 A schematic diagram of mechanism of exosomal HSPC111 facilitates CRLM via reprogramming lipid metabolism in CAFs. Exosomal HSPC111 derived from CRC cells phosphorylates ACLY in CAFs, leading to the increased levels of acetyl-CoA and histone acetylation to secrete CXCL5, and resulting in CRC cells colonized in liver via the CXCL5-CXCR2 axis.

Article Snippet: Cells were cultured in the presence of appropriate exosomes (20μg/mL) for 48 h before supernatants were collected, and the CXCL5 level was detected by CXCL5 ELISA kit (EK0728, BOSTER) according to the manufacturer’s protocols.

Techniques: Derivative Assay

Journal: Cell Discovery

Article Title: Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer

doi: 10.1038/s41421-024-00720-w

Figure Lengend Snippet: a Cytokine array analysis of CM from Panc02.shCtrl vs Panc02.sh ζ cells in 0% FBS culture for 72 h. b Left: WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) expression in Panc02.shCtrl and Panc02.sh ζ cells cultured in 10% FBS or 0% FBS medium for 48 h. Right: WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) in 3D cultured PANC-1.shCtrl and PANC-1.sh ζ cells treated with Gem (20 nM) vs vehicle for 48 h. c WB analysis of CXCL2, CXCL5, 14-3-3ζ, and GAPDH (sample processing controls) in PANC-1.shCtrl and PANC-1.shζ, and 14-3-3ζ-overexpressing PANC-1.sh ζ cells treated with Gem (20 nM) vs vehicle for 48 h. d WB analyses of Yap1, CXCL2, CXCL5, and GAPDH (sample processing controls) expression in Panc02.shCtrl and Panc02.sh Yap1 cells in 0% FBS culture for 24 h. Representative data of two independent repeats. e ChIP-qPCR assays of Yap1 binding to CXCL2/5 promoter region in 3D-cultured PANC-1 cells with 24 h of Gem (20 nM) treatment (mean ± SD, t -test, n = 3 biological repeats). f WB analysis of CXCL2, CXCL5, and GAPDH (sample processing controls) in Panc02.shCtrl and Panc02.sh NLK sublines cultured in 0.1% FBS for 24 h. Representative data of two independent repeats. g Schematics (left) and relative numbers of proliferating (middle) and apoptotic (right) cells from 3D-cultured PATC53 cells treated with CM from 3D-cultured hPSCs that were activated by adding CM from 8.5 nM Gem-treated 3D-cultured PATC53 cells plus CXCL2 (1 μg/mL) or CXCL5 (3 μg/mL) blocking antibodies for 48 h (mean ± SD, t -test, n = 3 biological repeats). h Schematics and relative cell number of 3D-cultured KPC mT3 cells treated with CM from 3D-cultured mPSCs added with vehicle, recombinant CXCL2 (0.5 ng/mL), or recombinant CXCL5 (0.1 µg/mL) proteins for 48 h (mean ± SD, t -test, n = 3 biological repeats). i Stress-induced 14-3-3ζ-Yap1-CXCL2/5 pathway in PDAC cells activates fibroblasts, which turns on the adaptive response that enables PDAC cells to survive under stress conditions.

Article Snippet: The blocking antibodies to mouse CXCL2 (MAB452), mouse CXCL5 (MAB433), human CXCL5 (MAB254), and control antibody (MAB0061) were purchased from R&D Systems and human CXCL2 (311001) was purchased from Biolegend (California, USA).

Techniques: Expressing, Cell Culture, Binding Assay, Blocking Assay, Recombinant

Journal: Cell Discovery

Article Title: Targeting a chemo-induced adaptive signaling circuit confers therapeutic vulnerabilities in pancreatic cancer

doi: 10.1038/s41421-024-00720-w

Figure Lengend Snippet: a WB analyses of Cox2 and GAPDH (sample processing controls) in mPSCs (3D culture) incubated with recombinant CXCL2 (0.5 ng/mL) or CXCL5 (0.1 µg/mL) for 24 h. Representative data of two independent repeats. b WB analysis of Cox2 and GAPDH (sample processing controls) in NIH3T3 cells treated with CM from Panc02.shCtrl, Panc02.sh CXCL2 , or Panc02.sh CXCL5 cells cultured in 0% FBS for 72 h. Representative data of two independent repeats. c PGE2 concentration in CM from NIH3T3 cells treated with recombinant CXCL2 (0.5 ng/mL) or CXCL5 (0.1 µg/mL) for 48 h (mean ± SD, t -test, n = 3 biological repeats). d Relative numbers of Panc02 cells treated with PGE2 in 0% FBS for 48 h (mean ± SD, t -test, n = 3 biological repeats). e Relative numbers of PANC-1 cells in upper chambers of Transwell units 3D-cocultured with hPSCs in lower chambers of Transwell units with or without Cel (4 nM) and Gem (20 nM) for 72 h (mean ± SD, t -test, n = 3 biological repeats). f Relative number of Panc02 cells treated with CM from WT mPSCs or Cox2 –/– mPSCs activated by CM from Panc02 cells in 0% FBS for 48 h (mean ± SD, t -test, n = 3 biological repeats). g RPPA analysis of NIH3T3 cells treated with CM collected from Panc02.shCtrl and Panc02.sh ζ cells cultured in 10% or 0% FBS medium. h WB analysis of pT346-NDRG1, NDRG1, and GAPDH (sample processing controls) in 3D-cultured hPSCs treated for 24 h with CM collected from 3D-cultured PANC-1.shCtrl and PANC-1.sh ζ cells treated with vehicle/Gem (20 nM) for 24 h. Representative data of two independent repeats. i WB analysis of pT346-NDRG1, NDRG1, pS473-Akt, Akt, and GAPDH (sample processing controls) expression in 3D-cultured hPSCs treated with recombinant CXCL2 (15 ng/mL) or CXCL5 (25 ng/mL) for 24 h. Representative data of two independent repeats. j WB analysis of Rictor, pT346-NDRG1, NDRG1, Cox2, and GAPDH (sample processing controls) protein in NIH3T3.shCtrl and NIH3T3.sh Rictor cells treated for 24 h with recombinant CXCL2 (0.5 ng/mL) or recombinant CXCL5 (0.1 µg/mL). Representative data of two independent repeats. k, l IHC staining of indicated proteins of PDAC tumor tissues from KPC and KPC -ζ fl/fl mice with indicated treatments (mean ± SD, t -test, n = 5 biological repeats, scale bar: 20 µm). m PDAC cells and fibroblasts together create the adaptive stress response circuit, which is essential for PDAC cell adaptation to stresses, including nutrient deprivation and chemotherapy-induced genotoxicity. Stressors promote the Yap1-CXCL2/5 signaling axis via NLK in 14-3-3ζ-overexpressing PDAC cells, leading to the activation of the CXCR2-mTORC2-Cox2-PGE2 pathway in fibroblasts. Reciprocally, PGE2 from fibroblasts promotes PDAC cell survival and adaptive resistance to Gem.

Article Snippet: The blocking antibodies to mouse CXCL2 (MAB452), mouse CXCL5 (MAB433), human CXCL5 (MAB254), and control antibody (MAB0061) were purchased from R&D Systems and human CXCL2 (311001) was purchased from Biolegend (California, USA).

Techniques: Incubation, Recombinant, Cell Culture, Concentration Assay, Expressing, Immunohistochemistry, Activation Assay

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: a , b Culture supernatants collected from MSCs on day 3 were analyzed using a Human XL Cytokine Array Kit. Several cytokines with differential expression were found in the culture supernatants of ASMSCs, including CXCL5, ANG, GROα, and THBS1. c Expression levels of these cytokines in ASMSCs and HDMSCs were confirmed by qRT-PCR. d Western blot analysis was performed to detect the protein level of CXCL5 in ASMSCs compared with HDMSCs. e Quantitative data for western blot analyses are shown. f CXCL5 secreted from HDMSCs and ASMSCs was measured by ELISA on day 3. Data are presented as the mean ± SD ( n = 30). The results represent three independent experiments. *, p < 0.05; HDMSCs, mesenchymal stem cells from healthy donors; ASMSCs, mesenchymal stem cells from patients with ankylosing spondylitis; ANG, angiogenin; THBS1, thrombospondin-1

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Quantitative Proteomics, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: CD14 + monocytes were cultured with HDMSCs or ASMSCs after knocking down CXCL5 in the presence of M-CSF and RANKL. a Secretion of CXCL5 from MSCs was detected by ELISA on day 3 after transfection with sh-CXCL5. b Representative images of TRAP staining (× 100). c The number of TRAP + osteoclasts in each well is shown. d Representative images of F-actin assays (× 200). e Representative images of bone resorption assays (× 200). f Pit formation on each slide was assessed. Data are presented as the mean ± SD ( n = 30). The results represent three independent experiments. *, p < 0.05; HDMSCs, mesenchymal stem cells from healthy donors; ASMSCs, mesenchymal stem cells from patients with ankylosing spondylitis

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Staining

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: a Representative images of TRAP staining (× 100), F-actin and bone resorption assays (× 200) of osteoclasts treated with the indicated doses of CXCL5 under osteoclastogenic conditions. b The number of TRAP + osteoclasts in each well on day 9 is shown. c Pit formation on each slide on day 15 was assessed. d mRNA expression levels of TRAP, CTSK, and NFATc1 were determined by qRT-PCR on day 9. e Protein levels of TRAP, CTSK, and NFATc1 were determined by western blot analysis on day 9. f Quantitative data for TRAP, CTSK, and NFATc1 protein levels determined by western blot analyses are shown. g Activation of signaling pathways involved in osteoclastogenesis was determined by western blot analyses on day 9. h Quantitative data for activation of signaling pathways determined by western blot analyses are shown. Data are presented as the mean ± SD ( n = 18). The results represent three independent experiments. *, p < 0.05

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Staining, Expressing, Quantitative RT-PCR, Western Blot, Activation Assay, Protein-Protein interactions

Journal: Cell Death & Disease

Article Title: Abnormal inhibition of osteoclastogenesis by mesenchymal stem cells through the miR-4284/CXCL5 axis in ankylosing spondylitis

doi: 10.1038/s41419-019-1448-x

Figure Lengend Snippet: a Bioinformatics analysis in three databases (TargetScan, miRBase, and miRDB) to identify possible miRNAs targeting CXCL5. Seven miRNAs with the highest scores for targeting CXCL5 are shown. b Expression levels of miRNAs in HDMSCs and ASMSCs were determined by qRT-PCR. c Expression levels of miR-4284 in HDMSCs and ASMSCs after treatment with miR-4284 inhibitor or mimic. d Expression levels of CXCL5 in HDMSCs and ASMSCs after treatment with miR-4284 inhibitor or mimic. e Possible binding sites between miR-4284 and CXCL5 are shown, and a mutant (MUT) CXCL5 site was constructed. f Binding sites were confirmed by luciferase assays. The miR-4284 mimic decreased the luciferase activity of wild-type (WT) CXCL5 but had no impact on MUT CXCL5. Data are presented as the mean ± SD ( n = 30). The results represent three independent experiments. *, p < 0.05; HDMSCs, mesenchymal stem cells from healthy donors; ASMSCs, mesenchymal stem cells from patients with ankylosing spondylitis

Article Snippet: The level of CXCL5 protein in the cell culture supernatant was quantitated using Human CXCL5 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Mutagenesis, Construct, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Adipose-Tumor Crosstalk contributes to CXCL5 Mediated Immune Evasion in PDAC

doi: 10.1101/2023.08.15.553432

Figure Lengend Snippet: : Schematic created in BioRender illustrating human adipose tissue conditioned media (hAT-CM). : Proliferation measured by EdU incorporation of cells in vitro in response to 24h of stimulus with pooled hAT-CM. : Kaplan-Meier Plot generated using GEPIA showing significant differences between the upper and lower quartile expressing patients in the PAAD-TCGA dataset. : Stimulation of cells with hAT-CM shows CXCL5 in the media of the stimulated cells measured by ELISA. Statistical analyses were performed using Brown-Forsythe and Welch corrected One-Way Anova in GraphPad Prism.

Article Snippet: The collected media was then measured for CXCL5 using a Human CXCL5/ENA-78 DuoSet ELISA kit [R&D Systems DY254] and DuoSet ELISA Ancillary Reagent Kit 2 [R&D Systems DY008B] according to the manufacturer’s protocol.

Techniques: In Vitro, Generated, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Adipose-Tumor Crosstalk contributes to CXCL5 Mediated Immune Evasion in PDAC

doi: 10.1101/2023.08.15.553432

Figure Lengend Snippet: hAT-CM of pooled obese samples or recombinant IL-1b, TNF, or combo (1 ug/mL each) was used to stimulate CXCL5 release. Neutralizing antibodies against IL-1b or TNF were applied at 1 ug/mL to inhibit CXCL5 secretion in the presence of hAT-CM. Cultures were treated for 36h. Statistics were performed by Brown-Forsythe & Welch’s One Way ANOVA.

Article Snippet: The collected media was then measured for CXCL5 using a Human CXCL5/ENA-78 DuoSet ELISA kit [R&D Systems DY254] and DuoSet ELISA Ancillary Reagent Kit 2 [R&D Systems DY008B] according to the manufacturer’s protocol.

Techniques: Recombinant

Journal: bioRxiv

Article Title: Adipose-Tumor Crosstalk contributes to CXCL5 Mediated Immune Evasion in PDAC

doi: 10.1101/2023.08.15.553432

Figure Lengend Snippet: (A) CXCL5 was validated for knockout in murine PDAC cell lines (KPC) by qPCR. Proliferation of the cells in vitro was measured by EdU incorporation over a 6h time period in 5% DMEM. (C-U) The knockout cells were pooled (3-15 + 3-17, or 45-4+45-15) and orthotopically injected into the pancreas of obese mice. Tumors were allowed to grow for 29 days prior to takedown when they were assessed for tumor weight and immune infiltration by flow cytometry (see Supplement 4 for gating strategy and antibodies). Statistics were performed by Brown-Forsythe & Welch’s One Way ANOVA (4B, 4D), or Welch’s T-test (4E-U).

Article Snippet: The collected media was then measured for CXCL5 using a Human CXCL5/ENA-78 DuoSet ELISA kit [R&D Systems DY254] and DuoSet ELISA Ancillary Reagent Kit 2 [R&D Systems DY008B] according to the manufacturer’s protocol.

Techniques: Knock-Out, In Vitro, Injection, Flow Cytometry

Journal: bioRxiv

Article Title: Adipose-Tumor Crosstalk contributes to CXCL5 Mediated Immune Evasion in PDAC

doi: 10.1101/2023.08.15.553432

Figure Lengend Snippet: Mice underwent diet-induced obesity and tumor implantation as in . At day 10 post tumor implantation, mice were treated with 200 ug of anti-PD-1 antibody, or IgG and continued every 3 rd day until d22. Pancreas weight was assessed at endpoint (B). Statistics were performed by Brown-Forsythe & Welch’s One Way ANOVA. (C) Adipose tissue contains cytokines that can stimulate CXCL5 release from PDAC cells, which acts as a critical deterrent to tumor T cell infiltration (figure created in Biorender).

Article Snippet: The collected media was then measured for CXCL5 using a Human CXCL5/ENA-78 DuoSet ELISA kit [R&D Systems DY254] and DuoSet ELISA Ancillary Reagent Kit 2 [R&D Systems DY008B] according to the manufacturer’s protocol.

Techniques: Tumor Implantation

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 1. CXCL5 protein expression is concordant with prostate cancer progression. Shown are representative panels from a hematox- ylin and eosin–stained, high-density tissue microarray probed with antibody against CXCL5, as follows: (A) Benign glands demonstrating weak staining. (B) PCa (Gleason sum 3 + 3) demonstrating weak staining. (C) PCa (Gleason sum 4 + 4) demonstrating moderate to strong staining. (D) Hormone refractory METs demonstrating strong staining. (E) PCa demonstrating moderate to strong staining asso- ciated with stromal inflammatory component (yellow arrows point to areas of inflammation). (F) Benign glands demonstrating strongly staining luminal secretions (black arrows). Original magnifications, ×100. Panel E has been enlarged further, ×4, to illustrate the area of inflammatory infiltrate concomitant with CXCL5 protein expression. (G) Boxplot depicting median product score distributions of protein expression levels for benign glands, malignant glands from PCa, and malignant areas from METs and P values associated with the statistical evaluation of these distributions.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Expressing, Staining, Microarray

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 2. Nontransformed and transformed prostate epithelial cells express the CXCL5 receptor and endogenously secrete CXCL5. (A) Immunoblot analysis of protein lysates prepared from transformed PC3 and LNCaP, and nontransformed N15C6 and BPH-1 prostate epithelial cells probed with antibodies specific for the CXCL5 receptor, CXCR2, and loading control, β-actin. Pri- mary antibody concentrations used were 1:1000 for CXCR2 and 1:5000 for β-actin. (B) Protein levels (pg/ml) of CXCL5 present in media conditioned by transformed LNCaP and PC3 or nontrans- formed N15C6 or BPH-1 cells prostate epithelial cells were deter- mined by ELISA. The graph shows the pg/ml CXCL5 detected plotted on a logarithmic scale (y axis).

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

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

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 4. CXCL5-stimulated proliferative and invasive responses. (A) N15C6 (light gray bars) or BPH-1 (dark gray bars) nontransformed prostate epithelial cells proliferated to significantly higher levels when grown for 72 hours in SF media supplemented with 10 pM CXCL5 than those grown in SF alone (*P < .001). Preincubation of the cells for 1 hour with 1 μg/ml antibody against CXCR2, the receptor for CXCL5, followed by supplementation with CXCL5 and maintenance of growth in CXCL5 + anti-CXCR2–containing media significantly ablated the proliferative response (#P < .001). In contrast, cellular growth after preincubation with an antibody against an unrelated chemokine receptor, CXCR4, followed by supplementation with CXCL5 and maintenance of growth in CXCL5 + anti-CXCR4–containing media was similar to that observed for non–pretreated cells grown in CXCL5-supplemented media and was significantly higher than that in SF alone (*P < .001). All data are shown normalized to growth in unsupplemented SF, which was set at one-fold. (B) N15C6 (LEFT) or LNCaP (RIGHT) cells were grown in SF media (untreated, UnT) or SF media supplemented with 10 pM CXCL5 for N15C6 or 100 pM CXCL5 for LNCaP (treated, T) for the times indicated. The cells were then harvested and assessed for nucleosomal DNA fragmentation. The fraction of cells exhibiting apoptosis plotted on the y axis was calculated as the difference in absorbance measured at 405 nm and at the reference wavelength of 490 nm after adjusting for background absorbance at both wavelengths. No significant differences in the fraction of cells exhibiting apoptosis were observed between treated and untreated cells at any time point, demonstrating that CXCL5 does not promote antiapoptotic responses in these cells. (C) Fifteen thousand each of N15C6 (black bars) or PC3 (gray bars) cells were plated onto Matrigel-coated membranes and were exposed to complete media or complete media supplemented with 20 nM CXCL5 for 24 hours. After 24 hours, the cells that migrated and invaded through the Matrigel were stained and counted. N15C6 cells did not demonstrate an invasive response to treatment with CXCL5. However, approximately six-fold more PC3 cells migrated through the synthetic basement membrane, Matrigel, in response to 20 nM CXCL5 compared to vehicle (control, set at one-fold) (*P < .05). PC3 cell invasion through the Matrigel in response to CXCL5 was significantly inhibited by pretreatment with 1 μg/ml blocking antibody (anti- CXCR2) (#P < .05) but not by pretreatment with nonspecific antibody (anti-CXCR4) (*P < .05).

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Staining, Membrane, Control, Blocking Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 5. CXCL5 activates MAPK signaling in nontransformed N15C6 prostate epithelial cells. Nontransformed N15C6 cells rapidly and transiently phosphorylated ERK 1/2 and STAT3 when treated with either subnanomolar (10 or 100 pM) or nanomolar (1 nM) levels of CXCL5, whereas NF-κB subunit activation was evident only after treatment with 1 nM CXCL5. Primary antibody concentrations used were 1:500 for phospho-ERK, 1:500 for phospho-65 (NF-κB), 1:1000 for phospho-STAT3, 1:1000 for total ERK, 1:1000 for total p65, and 1:2000 for total STAT3. A total of 20 μg of protein lysate was electrophoresed per well. Immunoblots are shown on the left, and corresponding densitometric evaluations of the same blots are shown on the right. Phosphorylation relative to total protein quantitated from the immunoblot is shown in the densitometric plots as phospho/total protein.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 6. CXCL5 activates both MAPK and PI3K signaling in transformed LNCaP prostate epithelial cells. Transformed LNCaP cells rapidly and transiently phosphorylated both ERK 1/2 and the p65 subunit of NF-κB on treatment with subnanomolar (10 or 100 pM) levels of CXCL5. Immunoblots are shown in the top panel, and corresponding densitometric evaluations of the same blots are shown in the bottom panel. Phosphorylation relative to total protein quantitated from the immunoblot is shown in the densitometric plots as phospho/total protein. A total of 100 μg of protein lysate was electrophoresed per well. Primary antibody concentrations used were as described for Figure 5.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Transformation Assay, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: CXCL5 promotes prostate cancer progression.

doi: 10.1593/neo.07976

Figure Lengend Snippet: Figure 7. CXCL5 stimulates a transcriptional response in both nontransformed and transformed prostate epithelial cells. Quantitative real-time PCR of RNA purified from N15C6 cells (left) or LNCaP cells (right) treated with subnanomolar CXCL5 as shown demonstrates rapid and robust transcription of the EGR1 gene significantly higher than levels obtained at time 0 (set at one-fold) (*P < .05). Data shown are averaged from three or more separate experiments per time point per concentration of CXCL5 examined.

Article Snippet: To assess the effects of exogenous CXCL5 on cellular proliferation, recombinant human CXCL5 (254-X; R&D Systems) was added at the desired concentration in SF media (alone for control) to each well.

Techniques: Transformation Assay, Real-time Polymerase Chain Reaction, Purification, Concentration Assay