Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Cancer-associated fibroblasts promote pro-tumor functions of neutrophils in pancreatic cancer via IL-8: potential suppression by pirfenidone

doi: 10.1007/s00262-025-03946-z

Figure Lengend Snippet: IL-8 secreted by activated neutrophils is suppressed by PFD. A IL-8 secretion by neutrophils measured by ELISA. IL-8 secretion was significantly increased in neutrophils stimulated with CAF-CM compared to non-stimulated neutrophils. PFD administration during neutrophil activation with CAF-CM significantly suppressed IL-8 secretion. B Effect of secretions from activated neutrophils on cancer cell proliferation. The presence of neutrophils significantly increased MIA PaCa-2 proliferation. Mean ± SD is shown. Each dot represents the number of cells per well. Student’s t-test was used. ***, P < 0.001. C Pancreatic cancer cells induce fibroblasts to become iCAFs, and iCAF-CM activates neutrophils, increasing their IL-8 secretion and enhancing the malignant traits of pancreatic cancer cells. Conversely, PFD acts on activated neutrophils, suppressing the malignant traits of pancreatic cancer cells. This figure was created with BioRender.com

Article Snippet: To evaluate IL-8 levels in neutrophil secretions, culture supernatants were assayed using a human IL-8 enzyme-linked immunosorbent assay (ELISA) kit (Proteintech, KE00006) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

doi: 10.3390/ijms19113530

Figure Lengend Snippet: CTSS increases IL-1β , IL-8 , IL-6 , and TNF-α gene expression after 2- and 4-hours of treatment in a human corneal epithelial cell line (HCE-T cells) ( A ) IL-1β gene expression without and with CTSS treatment in HCE-T cells; ( B ) IL-8 gene expression without and with CTSS treatment in HCE-T cells; ( C ) IL-6 gene expression without and with CTSS treatment in HCE-T cells; ( D ) TNF-α gene expression without and with CTSS treatment in HCE-T cells. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

Article Snippet: The human IL-8 ELISA kit was from Innovative Research (Novi, MI, USA).

Techniques: Expressing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

doi: 10.3390/ijms19113530

Figure Lengend Snippet: CTSS significantly increases IL-8, IL-6, and TNF-α, IL-1β protein expression in cell culture medium and cell lysates from human corneal epithelial cells (HCE-T cells) at 2, 4, and 8 h of exposure. ( A ) IL-8 protein expression in cell culture medium from HCE-T cells without and with CTSS; ( B ) IL-8 protein expression in cell lysates from HCE-T cells without and with CTSS; ( C ) IL-6 protein expression in cell culture medium from HCE-T cells without and with CTSS; ( D ) IL-6 protein expression in cell lysates from HCE-T cells without and with CTSS; ( E ) TNF-α protein expression in cell culture medium from HCE-T cells without and with CTSS; ( F ) TNF-α protein expression in cell lysates from HCE-T cells without and with CTSS; ( G ) IL-1β protein expression in cell culture medium from HCE-T cells without and with CTSS; ( H ) IL-1β protein expression in cell lysate from HCE-T cells without and with CTSS. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of proteins of interest were normalized to total protein concentration of respective cell culture medium or lysates. a = individual ELISA kit ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

Article Snippet: The human IL-8 ELISA kit was from Innovative Research (Novi, MI, USA).

Techniques: Expressing, Cell Culture, Activity Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

doi: 10.3390/ijms19113530

Figure Lengend Snippet: CTSS activity is required for early induction of pro-inflammatory cytokines in human corneal epithelial cells. ( A ) IL-8 gene expression in HCE-T cells without (untreated), with heat-inactivated CTSS, and with active CTSS; ( B ) IL-6 gene expression in HCE-T cells without (untreated), with heat-inactivated CTSS, and with active CTSS. IL-8 and IL-6 gene expression were normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, ** p ≤ 0.01, data are represented as mean ± SEM, and one-way ANOVA with Tukey’s multiple comparison was used to compare cells within different CTSS treatments. The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Heat inactivation was by heating at 90 °C for 30 min.

Article Snippet: The human IL-8 ELISA kit was from Innovative Research (Novi, MI, USA).

Techniques: Activity Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

doi: 10.3390/ijms19113530

Figure Lengend Snippet: Experimental design to probe the effect of CTSS activation of PAR-2 on the increase in pro-inflammatory cytokines (IL-8, IL-6, TNF-α, and IL-1β) and proteases (CTSS and MMP-9) at 4 and 24 h of recombinant CTSS treatment.

Article Snippet: The human IL-8 ELISA kit was from Innovative Research (Novi, MI, USA).

Techniques: Activation Assay, Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

doi: 10.3390/ijms19113530

Figure Lengend Snippet: Gene expression of pro-inflammatory cytokines ( IL-8 , IL-6 , TNF-α , and IL-1β ) after 15 min, 1 h, and 2 h of CTSS treatment in human corneal epithelial cells (HCE-T cells). The amount of CTSS added corresponded to an activity level found in the 90th–95th percentile of SS patients (18,000 RFU, added to 500 µL of cell medium), as described in detail in Methods . Expression of genes of interest was normalized to expression of the endogenous gene, GAPDH ( n = 3 samples/group, * p ≤ 0.05, data are represented as mean ± SEM and one-way ANOVA with Dunnett’s multiple comparison was used to compare treated to untreated cells).

Article Snippet: The human IL-8 ELISA kit was from Innovative Research (Novi, MI, USA).

Techniques: Expressing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Cathepsin S Alters the Expression of Pro-Inflammatory Cytokines and MMP-9, Partially through Protease—Activated Receptor-2, in Human Corneal Epithelial Cells

doi: 10.3390/ijms19113530

Figure Lengend Snippet: The possible cell signaling pathways induced by acute exposure to 4 h of CTSS in human corneal epithelial cells contributing to increased release of pro-inflammatory cytokines and MMP-9 that may contribute to ocular surface inflammation. ( A ) The effects of acute CTSS on increased pro-inflammatory cytokines and MMP-9 mediated by PAR-2; ( B ) Acute CTSS induces IL-8 through a PAR-2- independent pathway. A one-way black solid arrow ( ) represents a proposed inducible pathway, an one-way black dashed arrow ( ) represents a possible one-way inducible pathway, a two-way red dashed arrow ( ) represents a possible two-way inducible pathway and a solid line with blunt end ( ) represents a negative feedback pathway.

Article Snippet: The human IL-8 ELISA kit was from Innovative Research (Novi, MI, USA).

Techniques:

Journal: Cell reports

Article Title: Collagen 1-mediated CXCL1 secretion in tumor cells activates fibroblasts to promote radioresistance of esophageal cancer.

doi: 10.1016/j.celrep.2023.113270

Figure Lengend Snippet: Figure 4. CXCL1 promotes migration and activation of fibroblasts via the CXCR2-STAT3 pathway (A) Western blot analysis of myCAF- and iCAF-related markers in fibroblasts incubated with CM from CXCL1-knockdown ESCC cells. (B) Quantification of migration in (Figure S5B) (n = 3 biological replicates). (C) Western blot analysis of myCAF- and iCAF-related markers in fibroblasts treated with indicated concentration of rCXCL1 proteins for 24 h. (D) Representative images of fibroblasts migration caused by indicated concentration of rCXCL1 proteins. Scale bar, 100 mm. (E) Quantification of migration in (D) (n = 3 biological replicates). (F) Western blot analysis of CAF markers in fibroblasts pre-treated with SB-265610 for 6 h and then treated by CM of KYSE510 for 18 h. KYSE510 cells were treated with Col1 or not. (G) Western blot analysis of CAF markers in fibroblasts pre-treated with SB-265610 for 6 h and then treated by rCXCL1 for 18 h. For all panels, data are presented as mean ± SD. **p < 0.01, ***p < 0.001, and ****p < 0.0001, as determined using two-tailed Student’s t test. Each assay for western blot had three biological repeats, and the quantification results are presented below each band. See also Figure S5.

Article Snippet: For CXCR2 receptor inhibitory assay, CXCR2 inhibitor SB-265610 (MCE, HY-50688) was added to serum-free medium for 6 h after fibroblasts grew until confluency, then CXCL1 recombinant protein was added to medium for another 18 h.

Techniques: Migration, Activation Assay, Western Blot, Incubation, Knockdown, Concentration Assay, Two Tailed Test