Journal: Molecular Medicine

Article Title: Metformin alleviates the calcification of aortic valve interstitial cells through activating the PI3K/AKT pathway in an AMPK dependent way

doi: 10.1186/s10020-021-00416-x

Figure Lengend Snippet: Metformin alleviates aortic calcification by activating the PI3K/AKT signaling pathway in a concentration-dependent manner in vitro. a Metformin inhibits inflammatory factor secretion including IL6, IL8, and MCP-1 in cell supernatant, after treatment with phosphate medium (PM) with or without metformin for 72 h as determined by ELISA; b ROS production was detected and quantified after PM treatment with or without metformin for 72 h; c Cell viability was detected by CCK8 after treatment with PM with or without metformin for 72 h; d Immunofluorescence staining images of p-AKT and OPN expression in AVICs after PM treatment with or without 100 μM metformin for 72 h, Scale bar, 50 μm; e The protein expression of p-AMPK (Thr172), p-AKT (Ser473), PI3K, p-eNOS (Ser1177), BMP2, and OPN in AVICs after PM treatment with or without metformin for 72 h as determined by WB; f Calcium deposition were detected by ARS Staining after various treatments for 7 days, Scale bar, 200 μm. n = 6 per group. CTL, control; Met, metformin; *p < 0.05 versus PM group (one-way ANOVA with Bonferroni post hoc test)

Article Snippet: The levels of IL6 (KE00007, Proteintech, China), IL8 (KE00006, Proteintech, China), and MCP-1 (KE00091, Proteintech, China) in the supernatant of AVICs were determined by ELISA.

Techniques: Concentration Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing, Control

Journal: Cell Death & Disease

Article Title: Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy

doi: 10.1038/s41419-023-06230-z

Figure Lengend Snippet: A Heatmap showing the transcriptomic expression levels of the secretory proteins in 10 NA and 10 CAA tissues. B Volcano plots depict DEGs of secretory proteins. C , D GO and KEGG enrichment scatter plot for upregulated transcriptome genes for the secretome in CAAs compared with NAs. E , F GSVA and GSEA pathway enrichment analysis of upregulated transcriptome genes for the secretome. G Heatmap of the top 20 differentially expressed transcriptome genes in the secretome. H ELISAs detected 11 secretory proteins in NA and CAA supernatants. I Two overlapping sets with Veen diagrams of upregulated secretory proteins between RNA-seq and ELISA. J , K IHC and IF staining of CXCL8 in 10 NA and 10 CAA tissues. L IF staining of NAs and CAAs. M , N CXCL8 gene and protein expression in MDA-MB-231 cells after coculture without or with NAs or CAAs for 7 days. O GSEA of the “INTERLEUKIN-8_PRODUCTION” gene set in the CAAs versus NAs. P Overall survival analysis of independent breast cancer cohort GSE20685 for the prognostic potential of CXCL8. CAA cancer-associated adipocyte, CXCL8 C-X-C motif chemokine ligand 8, DEGs differentially expressed genes, ELISA enzyme-linked immunosorbent assay, GSEA gene set enrichment analysis, GSVA gene set variation analysis, IF immunofluorescence, IHC immunohistochemistry, NA normal adipocyte. Data are presented as the means ± SD of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: MDA-MB-231 and 4T1 cells were treated with different concentration gradients of human CXCL8 (Cat #94853, CST) and recombinant mouse MIP2 (Cat#ab243752, Abcam) for 1 week, respectively.

Techniques: Expressing, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy

doi: 10.1038/s41419-023-06230-z

Figure Lengend Snippet: A Viability of MDA-MB-231 cells treated with different concentrations of CXCL8. B , C Representative images of wound healing and Transwell assays of MDA-MB-231 cells after treatment with CXCL8 (10 ng/ml) or CXCL8 (10 ng/ml) + CXCL8 antibody (10 μg/ml) for 7 days. D Viability of 4T1 cells treated with different concentrations of MIP2. E , F Representative images of wound healing and transwell assays of 4T1 cells after treatment with MIP2 (20 ng/ml) or CXCL8 (20 ng/ml) + MIP2 antibody (10 μg/ml) for 7 days. G , H Western blot and IF analysis of the EMT-related markers AKT and p-AKT in MDA-MB-231 cells after treatment with CXCL8 (10 ng/ml) or CXCL8 (10 ng/ml) + CXCL8 antibody (10 μg/ml) or CXCL8 (10 ng/ml) + GSK2141795 (10 μmol/L) for 7 days. I , J Western blot and IF analysis of the EMT-related markers AKT and p-AKT in MDA-MB-231 cells after treatment with MIP2 (20 ng/ml) or MIP2 (20 ng/ml) + MIP2 antibody (20 μg/ml) or MIP2 (10 ng/ml) + GSK2141795 (10 μmol/L) for 7 days. CXCL8 C-X-C motif chemokine ligand 8, DEGs differentially expressed genes, ELISA enzyme-linked immunosorbent assay, EMT epithelial–mesenchymal transition, GSEA gene set enrichment analysis, GSVA gene set variation analysis, IF immunofluorescence, IHC immunohistochemistry, MIP2 macrophage inflammatory protein-2, TNBC triple-negative breast cancer. Data are presented as the means ± SD of at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: MDA-MB-231 and 4T1 cells were treated with different concentration gradients of human CXCL8 (Cat #94853, CST) and recombinant mouse MIP2 (Cat#ab243752, Abcam) for 1 week, respectively.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Targeting cancer-associated adipocyte-derived CXCL8 inhibits triple-negative breast cancer progression and enhances the efficacy of anti-PD-1 immunotherapy

doi: 10.1038/s41419-023-06230-z

Figure Lengend Snippet: In TNBC, CAA is positively associated with an immunosuppressive microenvironment. CAA is highly expressed and secretes CXCL8, which promotes tumor growth and lung metastasis and regulates EMT through the PI3K/AKT pathway. CXCL8 modulated the immunosuppressive microenvironment by upregulating CD274 expression and suppressing T-cell infiltration. Blockade of the CAA-derived CXCL8 pathway can sensitize the immunotherapy response of PD-1, thus inhibiting TNBC progression. CAA cancer-associated adipocyte, NA normal adipocyte, PD-1 programmed cell death protein 1, CD274 programmed death-ligand 1.

Article Snippet: MDA-MB-231 and 4T1 cells were treated with different concentration gradients of human CXCL8 (Cat #94853, CST) and recombinant mouse MIP2 (Cat#ab243752, Abcam) for 1 week, respectively.

Techniques: Expressing, Derivative 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