Journal: European Journal of Medical Research

Article Title: PEAK1 promotes prostate cancer progression and docetaxel resistance by mediating the polarization of tumor-associated macrophages

doi: 10.1186/s40001-025-03568-2

Figure Lengend Snippet: Association of PEAK1 level and “M2” polarization of TAM in PCa. A , B Level of immune cell infiltration between the high and low PEAK1 expression groups. C–H Gene correlation analysis between PEAK1 and IL-10, TGF- β , CCL2, Arg1, CD163, and CD206 in PRAD was conducted via GEPIA ( http://gepia.cancer-pku.cn/ ). I IHC was conducted to detect CCL2 in the tumor tissues. Scale bar = 50 μm. J , K Western blotting was conducted to measure CCL2, Arg1, CD163, and CD206 in the tumor tissues. L IHC was conducted to detect CD45, iNOS, CD163, CD8, PD-L1, granzyme B, and IFN-γ in the tumor tissues. Scale bar = 50 μm. N = 3. * stands for p < 0.05, ** stands for p < 0.01, *** stands for p < 0.001

Article Snippet: Next, the levels of CCL2 and IL-6 in the cell supernatant were determined via CCL2 (#EK187EG) and IL-6 (#EK106) kits (Multi Sciences, Hangzhou, China), respectively.

Techniques: Expressing, Western Blot

Journal: European Journal of Medical Research

Article Title: PEAK1 promotes prostate cancer progression and docetaxel resistance by mediating the polarization of tumor-associated macrophages

doi: 10.1186/s40001-025-03568-2

Figure Lengend Snippet: Functions of PCa cells with PEAK1 upregulation on “M2” polarization of macrophages. A Co-culture system graph. THP1 cells were put in the lower chamber and PC3 or DU145 cells were put in the upper chamber. B RT-PCR was conducted to detect CCL2 and IL-6 mRNA expression in PC3 and DU145 cells. C , D ELISA was conducted to test the levels of CCL2 and IL-6 in the co-culture medium. E RT-PCR was conducted to detect the expressions of “M2” markers, including IL-10, TGF- β , CCL2, Arg1, CD163, and CD206 in THP1 cells co-cultured with PEAK1-overexpressed PCa cells. F Migration of THP1 cells was evaluated using a transwell assay. G , H RT-PCR and WB analysis of CCR2 expression in THP1 cells co-cultured with PEAK1-overexpressed or normal PC3/DU145 cells. I To test the functions of “M2” macrophages on the PCa cells, the THP1 cells were put in the upper transwell chamber, while the PC3 cells were put in the lower chamber. J CCK8 assay was performed to detect the viability of PC3 cells. K EdU-staining assay was performed to evaluate the proliferation of PC3 cells. The EdU-positive cell rate was counted. L Transwell assay was carried out to evaluate cell migration. M WB analysis for detecting E -cad, Vim, Snail, and Twist1. N CCK-8 assay was carried out to determine the IC₅₀ value of DTX in PC3 cells. O Colony formation assays were performed to determine the colony-forming ability of cells with DTX treatment. P WB analysis for assessing the expressions of Bcl-2, Bax and cleaved caspase-3 (c-Casp3). N = 3. Q , R PC3 cells and DU145 cells were, respectively, treated with TGF- β (10 ng/mL) or LY2157299 (10 μM), a selective TGF- β receptor type I (TGF–βRI) kinase inhibitor. RT-PCR and WB were conducted to detect the alteration of CCR2 expression in the two cells. * stands for p < 0.05, ** stands for p < 0.01, *** stands for p < 0.001

Article Snippet: Next, the levels of CCL2 and IL-6 in the cell supernatant were determined via CCL2 (#EK187EG) and IL-6 (#EK106) kits (Multi Sciences, Hangzhou, China), respectively.

Techniques: Co-Culture Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Migration, Transwell Assay, CCK-8 Assay, Staining

Journal: European Journal of Medical Research

Article Title: PEAK1 promotes prostate cancer progression and docetaxel resistance by mediating the polarization of tumor-associated macrophages

doi: 10.1186/s40001-025-03568-2

Figure Lengend Snippet: Mechanistic diagram of PEAK1 in PCa. Summary diagram of the functions and mechanisms of PEAK1 in PCa. Upregulated PEAK1 promotes the HIF-1α/STAT3/NF-κB pathway activation, thus enhancing tumor proliferation, migration, EMT, and reducing apoptosis, docetaxel/enzalutamide sensitivity. PEAK1 enhances CCL2, IL-6 and PD-L1 expression from PCa cells, which mediate “M2” polarization of TAMs and inducing TGF- β expression from TAMs. TGF- β can act on PCa cells and induce PEAK1 upregulation

Article Snippet: Next, the levels of CCL2 and IL-6 in the cell supernatant were determined via CCL2 (#EK187EG) and IL-6 (#EK106) kits (Multi Sciences, Hangzhou, China), respectively.

Techniques: Activation Assay, Migration, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: C1q/ TNF ‐related protein 9 inhibits the cholesterol‐induced Vascular smooth muscle cell phenotype switch and cell dysfunction by activating AMP‐dependent kinase

doi: 10.1111/jcmm.13196

Figure Lengend Snippet: CTRP 9 treatment alleviates cholesterol loading‐induced VSMC pro‐inflammatory factor secretion ( A ) Cell viability of VSMC s, measured using CCK 8, after incubation with CTRP 9 for 72 hrs. ( B ‐ D ) VSMC s were treated for 72 hrs with different concentrations of CTRP 9 (0–10 μg/ml) in the presence of 5 μg/ml cholesterol or were left untreated (control) before measurement of ( B ) IL ‐6 secretion, ( C ) IL ‐1β secretion and ( D ) MCP ‐1 secretion. Values represent the means ± S.D. of triplicate reactions and are representative of three independent experiments. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Recombinant human CTRP9 (6537‐TN‐050) and all ELISA‐based detection systems (IL‐6, VAL102, IL‐1β, VAL101 and MCP‐1, DCP00) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: CCK-8 Assay, Incubation

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 1. Identifi cation of culture conditions that enhance long-term CLL cell survival. Freshly isolated mononuclear cells were purifi ed on Ficol before culturing in RPMI supplemented with 10% heat inactivated FCS at a cell concentration of 1 – 60 10 6 cells/mL. (A) A single patient sample was seeded at the indicated densities and cell survival followed till day 14 ( * p 0.0001). (B) PBMCs were cultured at 5 10 7 cells/mL for 4 weeks with 50% medium changes every 2 – 3 days, weekly or fortnightly. (C) CLL PBMCs were cultured in complete medium for 7 days before supernatants were incubated with the ChemiArray III membrane. Complete medium was used as a negative control to account for non-specifi c binding. Seven CLL patient-samples were tested with similar results. Supernatant was collected from CLL PBMC cultures at indicated time points and (D) CCL2 and (E) CXCL2 protein concentration determined using ELISA. Results are displayed as mean SD ( n 2).

Article Snippet: A human CCL2/MCP-1 DuoSet ELISA Kit was used to detect CCL2 in the culture supernatant and a Human CCL2/MCP-1 Quantikine ELISA Kit to detect plasma CCL2 (both from R&D Systems), a Human Growth Regulated Oncogene beta (MIP-2, CXCL2) ELISA Development Kit (Promokine, Germany) was used to detect protein from plasma and culture supernatants, and Human IL-6 and Human IL-8 optEIA ELISA kits were used to detect protein from plasma (both from BD), all performed according to the manufacturers ’ instructions.

Techniques: Isolation, Concentration Assay, Cell Culture, Incubation, Membrane, Negative Control, Binding Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 3. Eff ect of IL-6, IL-8, CXCL2 and CCL2 on CLL cell survival in vitro . (A) CLL PBMCs were cultured for 7 days either alone or in the presence of recombinant human IL-6 (2.5 ng/mL) or IL-8 (2.5 ng/mL). Data are displayed as mean SD ( n 5) and each experiment was performed in triplicate ( * p 0.05, * * p 0.01). (B) CLL PBMCs were cultured with the addition of recombinant human CXCL2 (3.0 ng/mL) or CCL2 (2.5 ng/mL). Data are displayed as mean SD ( n 5) and each experiment was performed in triplicate ( * p 0.05, * * p 0.01). (C) B cells isolated from healthy donors were incubated with CCL2 and CXCL2 as described above. (D) Measurement of cell viability by trypan blue exclusion following the addition of recombinant human IL-6, IL-8, CCL2 or CXCL2 in diff erent combinations. Data are represented as mean fold-change SD ( n 6, each performed in duplicate or triplicate; * p 0.05, * * p 0.01).

Article Snippet: A human CCL2/MCP-1 DuoSet ELISA Kit was used to detect CCL2 in the culture supernatant and a Human CCL2/MCP-1 Quantikine ELISA Kit to detect plasma CCL2 (both from R&D Systems), a Human Growth Regulated Oncogene beta (MIP-2, CXCL2) ELISA Development Kit (Promokine, Germany) was used to detect protein from plasma and culture supernatants, and Human IL-6 and Human IL-8 optEIA ELISA kits were used to detect protein from plasma (both from BD), all performed according to the manufacturers ’ instructions.

Techniques: In Vitro, Cell Culture, Recombinant, Isolation, Incubation

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 4. Mechanism of chemokine eff ect in CLL in vitro culture. (A) CLL PBMCs were cultured in the presence of CCL2 (2.5 ng/mL) or CXCL2 (3 ng/mL), with or without the addition of anti-human CCL2 antibody, anti-human CXCL1/2/3/GRO pan-specifi c antibody, and/or an isoptype control antibody, all at a concentration of 1 μ g/mL. Data are displayed as mean SD and are representative of one patient, performed in triplicate. (B) Cell viability was examined after 14 days following weekly addition of CXCL2 and/or CCL2 or isotype control antibody (all at 1 μ g/mL). Data are displayed as mean SD ( n 5, each performed in duplicate or triplicate; * p 0.05). CLL PBMCs or CD19 selected CLL cells were examined for (C) CCL2 and (D) CXCL2 protein expression after 1 day and 1 week using ELISA. Data are displayed as mean SD ( n 2, each in triplicate; * p 0.05, * * p 0.01, * * * p 0.001).

Article Snippet: A human CCL2/MCP-1 DuoSet ELISA Kit was used to detect CCL2 in the culture supernatant and a Human CCL2/MCP-1 Quantikine ELISA Kit to detect plasma CCL2 (both from R&D Systems), a Human Growth Regulated Oncogene beta (MIP-2, CXCL2) ELISA Development Kit (Promokine, Germany) was used to detect protein from plasma and culture supernatants, and Human IL-6 and Human IL-8 optEIA ELISA kits were used to detect protein from plasma (both from BD), all performed according to the manufacturers ’ instructions.

Techniques: In Vitro, Cell Culture, Control, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 5. Confocal microscopy detects CCL2 and CXCL2 in cultured CLL PBMCs. Macrophage lineage cells and T cells were examined in 14-day-old CLL PBMC cultures for the production of CCL2 and CXCL2 by immunofl uorescence staining of CD68 and CD3 (magnifi cation 63). CD68 positive macrophage cells were examined for (A) CXCL2 and (B) CCL2, respectively, and CD3 positive T cells were examined for (C) CXCL2 and (D) CCL2, respectively. Cells staining positive for CD68 and CD3 appear red, cells staining positive for CCL2 and CXCL2 appear green, and nuclear staining appears blue (DAPI).

Article Snippet: A human CCL2/MCP-1 DuoSet ELISA Kit was used to detect CCL2 in the culture supernatant and a Human CCL2/MCP-1 Quantikine ELISA Kit to detect plasma CCL2 (both from R&D Systems), a Human Growth Regulated Oncogene beta (MIP-2, CXCL2) ELISA Development Kit (Promokine, Germany) was used to detect protein from plasma and culture supernatants, and Human IL-6 and Human IL-8 optEIA ELISA kits were used to detect protein from plasma (both from BD), all performed according to the manufacturers ’ instructions.

Techniques: Confocal Microscopy, Cell Culture, Staining

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 6. Accessory cells protect CLL cells from apoptosis in vitro . (A) CLL PBMCs or CD19 purifi ed CLL cells were cultured as described previously and cell viability determined after 14 days. Data are displayed as mean SD ( n 5, each performed in duplicate or triplicate; * * * p 0.001). (B) Cell viability was examined after 14 days on cultured CLL PBMCs or indicated cell subsets. Data shown are mean SD ( n 5, each performed in duplicate or triplicate; * p 0.05, * * p 0.01, * * * p 0.001). (C) CLL PBMCs or CD19 selected CLL cells were cultured in the presence of recombinant human CXCL2 (2.5 ng/mL) or CCL2 (2.5 ng/mL) and cell viability determined after 14 days. Data shown are mean SD ( n 4; * p 0.05). (D) CD19 selected cells were cultured either alone or in the presence of accessory cells (either co-cultured or separated by a transwell) and cell viability determined after 14 days using trypan blue exclusion. Data are displayed as mean SD ( n 3) and each experiment was performed in triplicate ( * * p 0.01, * * * p 0.001).

Article Snippet: A human CCL2/MCP-1 DuoSet ELISA Kit was used to detect CCL2 in the culture supernatant and a Human CCL2/MCP-1 Quantikine ELISA Kit to detect plasma CCL2 (both from R&D Systems), a Human Growth Regulated Oncogene beta (MIP-2, CXCL2) ELISA Development Kit (Promokine, Germany) was used to detect protein from plasma and culture supernatants, and Human IL-6 and Human IL-8 optEIA ELISA kits were used to detect protein from plasma (both from BD), all performed according to the manufacturers ’ instructions.

Techniques: In Vitro, Cell Culture, Recombinant

Journal: Journal for Immunotherapy of Cancer

Article Title: Disruption of tumor-intrinsic PGAM5 increases anti-PD-1 efficacy through the CCL2 signaling pathway

doi: 10.1136/jitc-2024-009993

Figure Lengend Snippet: PGAM5 reduced CCL2 secretion in HCC cells by downregulating DRP1. ( A ) Schematic diagram of the in vitro co-culture system for Huh7 shctrl or shPGAM5 cells and phorbol-12-myristate-13-acetate-induced THP-1 cells. ( B ) Quantitative PCR analysis of macrophage polarization maker genes of THP-1 cells co-cultured with indicated HCC cells. ( C ) cytokine antibody array incubated with culture medium of Huh7 shctrl or shPGAM5 cells. ( D ) Quantitative PCR analysis of macrophage M1/M2 polarization maker genes of THP-1 in the co-culture system, culture medium of Huh7 shPGAM5 cells were supplemented with recombinant CCL2 and HCCLM3 PGAM5 OE cells were supplemented with neutralizing-CCL2 antibody. ( E ) TOP20 GO enrichment of differentially expressed proteins in Huh7 shPGAM5 cells compared with shctrl cells. ( F ) Volcano plot of significantly differentially expressed proteins in Huh7 shPGAM5 cells compared with shctrl cells. ( G ) Western blot analysis of protein expression of PGAM5 and mitochondria dynamic proteins DRP1, MFN1, MFN2, OPA1 in HCC cells. ( H ) Western blot analysis of DRP1 overexpression efficiency in Huh7 shPGAM5 cells by lenti-virus infection. ( I ) Concentration of CCL2 in the supernatant of indicated HCC cells was measured by ELISA. ( J ) Western blot analysis of DRP1 silencing efficiency in HCCLM3 PGAM5-OE cells by transient transfection of SiRNA. ( K ) Concentration of CCL2 in the supernatant of indicated HCC cells was measured by ELISA kit. *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001, ns: not significant. HCC, hepatocellular carcinoma; IHC, immunohistochemistry; UMAP, uniform manifold approximation and projection.

Article Snippet: Concentrations of CCL2 in the supernatant were examined by ELISA kit (CSB-E04655h, cusabio) following manufacturer’s instructions.

Techniques: In Vitro, Co-Culture Assay, Real-time Polymerase Chain Reaction, Cell Culture, Ab Array, Incubation, Recombinant, Western Blot, Expressing, Over Expression, Virus, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Immunohistochemistry

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Construction and bioactivity evaluation of WEV-bFGF. ( A ) Spatial structure prediction of WEV-bFGF recombinant protein and natural bFGF; ( B ) SDS-PAGE of bFGF, KIT-bFGF and WEV-bFGF; ( C ) Western blot of native bFGF, KIT-bFGF and WEV-bFGF; ( D ) The biological activities of native bFGF, KIT-bFGF and WEV-bFGF by CCK-8 assay; ( E ) The fluorescence distribution of Delight 800 labeled recombinant proteins in hypoxic HK-2 cells; ( F ) Statistical analysis of fluorescence intensity of Dylight 800 in hypoxic HK-2 cells; Scale bar = 25 μm; ( G ) The protective effects of recombinant proteins on HK-2 cells after hypoxic injury by the CCK8 assay. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Recombinant, SDS Page, Western Blot, CCK-8 Assay, Fluorescence, Labeling

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: The evaluation of targeting capacity of WEV-bFGF in ischemic kidney in vivo . ( A ) At 6 h after administration, the in vivo fluorescence distribution of Dylight800-labeled recombinant proteins in major organs of AKI-injured rats by the small animal imaging system; ( B ) Western blot showing bFGF protein expression in ischemic renal tissue 6 h after administration; ( C ) Quantitative analysis of relative bFGF protein expression at 6 h; ( D ) The content of bFGF in ischemic kidneys by ELISA assay at 6 h after administration; ( E ) The content of bFGF in serum at 6 h after administration; ( F ) Immunofluorescence colocalization of bFGF and KIM-1 in the frozen sections at 6 h after administration, Scale bar = 50 μm; ( G ) Colocalization analysis of PBS, bFGF, KIT-bFGF and WEV-bFGF groups at 6 h after administration; ( H ) At 24 h after administration, the in vivo fluorescence distribution of Dylight800-labeled recombinant proteins in major organs of AKI-injured rats by the small animal imaging system; ( I ) Western blot showing bFGF protein expression in ischemic renal tissue 24 h after administration; ( J ) Quantitative analysis of relative bFGF protein expression at 24 h; ( K ) The content of bFGF in ischemic kidneys by ELISA assay at 24 h after administration; ( L ) The content of bFGF in serum at 24 h after administration; ( M ) Immunofluorescence staining showing colocalization of bFGF and KIM-1 at 24 h after administration, scale bar = 50 μm; ( N ) Colocalization analysis of PBS, bFGF, KIT-bFGF and WEV-bFGF groups at 24 h after administration. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 8.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: In Vivo, Fluorescence, Labeling, Recombinant, Imaging, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Functional tests and morphological analysis of the kidneys. ( A ) Experimental design of WEV-bFGF for the treatment of I/R; ( B ) Statistical analysis of serum Scr content for renal function evaluation at 24 h after renal I/R injury in rats; ( C ) Statistical analysis of serum Scr content for renal function evaluation at 24 h after renal I/R injury in rats; ( D ) H&E staining for kidney histopathological evaluation after I/R injury. Arrows indicated the sites of renal tubular injury. Scale bar = 20 μm; ( E ) Statistical analysis of renal tubular injury score; ( F ) MASSON staining for renal tissue fibrosis evaluation after I/R injury. Scale bar = 50 μm; ( G ) Statistical analysis of renal tissue fibrosis. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 8.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Functional Assay, Staining

Journal: Regenerative Biomaterials

Article Title: AI-guided design and optimization of a novel KIM-1-targeted peptide for bFGF delivery in acute kidney injury repair

doi: 10.1093/rb/rbag050

Figure Lengend Snippet: Transcriptome analysis of potential mechanism in WEV-bFGF mediating ischemic renal repair. ( A ) Volcano plots of gene differences between the WEV group and the PBS group; ( B ) KEGG analysis between the WEV group and the PBS group; ( C ) Volcano plots of gene differences between the WEV-BFGF group and the bFGF group; ( D ) KEGG analysis between the WEV-bFGF group and the bFGF group; ( E ) Heat map of differentially expressed genes related to repair and regeneration of AKI; ( F ) Quantitative PCR validation of key gene expression. All quantitative data are presented as mean ± SD, * P < 0.05, ** P < 0.01, N = 6.

Article Snippet: Subsequently, in accordance with the instructions of the Human bFGF ELISA Kit (EK0441, Boster, China), the protein extracted from kidney tissue and the content of bFGF in serum were determined.

Techniques: Real-time Polymerase Chain Reaction, Biomarker Discovery, Gene Expression