Journal: Non-coding RNA Research

Article Title: Aerobic exercise alleviates allergic airway inflammation by suppressing circMETTL9 -mediated formation of macrophage extracellular traps ⋆

doi: 10.1016/j.ncrna.2025.08.008

Figure Lengend Snippet: Communication Between Airway Epithelial Cells and Macrophages Mediated by CXCL12-CXCR4 Regulates METs. (A) The co-expression network of IGFBP3 and macrophage chemokines was predicted by the STRING database. (B) The binding of CXCL12 to the CXCR4 receptor was predicted in the CellphoneDB database. (C) Representative SYTOX Green staining in macrophages treated with or without CXCL12 (n = 3). (D) The protein expression of MPO and CitH3 in macrophages was detected by Western blot analysis (n = 3). (E – G) ELISA was used to detect the expression of CXCL12 in the cell supernatant of the co-culture system (n = 3). RT-qPCR was used to detect the expression of CXCL12 in BEAS-2B cells and CXCR4 in macrophages (n = 3). All data are expressed as means ± SD. ∗ P < 0.05. GAPDH was used as a loading control for all Western blot assays. All data are expressed as means ± SD. ∗ P < 0.05.

Article Snippet: To assess the expression levels of CXCL12, ELISA kits specifically for mouse CXCL12 (catalog #KE10049, Proteintech, Chicago, USA), and for human CXCL12 ELISA Kit (catalog #RK00266, ABclonal, China) were utilized according to the manufacturer's protocols.

Techniques: Expressing, Binding Assay, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Quantitative RT-PCR, Control

Journal: Non-coding RNA Research

Article Title: Aerobic exercise alleviates allergic airway inflammation by suppressing circMETTL9 -mediated formation of macrophage extracellular traps ⋆

doi: 10.1016/j.ncrna.2025.08.008

Figure Lengend Snippet: Overexpression of CircMETTL9 Counteracts the Reduction Effect of Aerobic Exercise on METs. (A) Schematic timeline of the experimental protocol. Day 0: AAV-LUNG-OE- circMETTL9 by the nasal drip. Days 14, 28, 42, and 56 represent intraperitoneal (i.p.) injections of OVA. Days 35–68 represent exposure to ovalbumin aerosol. Aerobic exercise adaptation occurred from days 35–37, and days 39 and 67 represent the initial and final physical tests. Aerobic exercise was initiated on day 42 and ended on day 66. Euthanasia was performed on day 70. (B) The expression of circMETTL9 was detected by RNA FISH staining (n = 6). (C) The expression of circMETTL9 was performed by RT-qPCR (n = 6). (D) ELISA was used to detect the expression of CXCL12 in the BALF (n = 6). (E) The mRNA expression of CXCL12 and CXCR4 was detected by RT-qPCR (n = 6). (F) Western blot analysis was used to detect the protein expression of CitH3 and MPO in the lung tissue (n = 6). (G) Representative immunofluorescence images of CitH3, MPO, and CD68 staining of lung tissues. (H) The result of the Western blot showed the effect of circMETTL9 overexpression on IGFBP3 and EIF4A3 expression (n = 6). A, OVA-induced asthmatic mice and infected with blank AAV; E, mice were subjected to aerobic exercise and infected with blank AAV; OE-A, OVA-induced asthmatic mice and infected with AAV overexpressing circMETTL9 ; OE-AE, OVA-induced asthmatic mice performed aerobic exercise and infected with AAV overexpressing circMETTL9 . All data were shown as the means ± SDs and were assessed by a paired two-tailed t -test. ∗ P < 0.05.

Article Snippet: To assess the expression levels of CXCL12, ELISA kits specifically for mouse CXCL12 (catalog #KE10049, Proteintech, Chicago, USA), and for human CXCL12 ELISA Kit (catalog #RK00266, ABclonal, China) were utilized according to the manufacturer's protocols.

Techniques: Over Expression, Aerosol, Expressing, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Infection, Two Tailed Test

Journal: Journal of Biomedical Research

Article Title: DEC1 promotes breast cancer bone metastasis through transcriptional activation of CXCR4

doi: 10.7555/JBR.39.20250031

Figure Lengend Snippet: DEC1 deficiency prevented breast bone metastasis induced by intracardiac injections of 4T1 cells via decreasing CXCR4/CXCL12 in mice. Forty 4-month-old mice, comprising twenty Dec1 +/+ and twenty Dec1 −/− , were divided into four groups: Dec1 +/+ -PBS, Dec1 +/+ -4T1, Dec1 −/− -PBS, and Dec1 −/− -4T1. Mice in the Dec1 +/+ -4T1 and Dec1 −/− -4T1 groups received an intracardiac injection of 4T1 mouse breast cancer cells to induce breast bone metastasis, while mice in the Dec1 +/+ -PBS and Dec1 −/− -PBS groups received an equal volume of PBS via the same method for two months. n = 10 for each group. A: Osteolytic bone injury in the four groups of mice by X-ray. B: The serum CXCL12 amount in the four groups of mice ( n = 3–5). C–F: The CA153, CK8, and Ki67 expression in the four groups of mice by immunohistochemical staining ( n = 3). G and H: The TRAP-positive cells in the four groups of mice ( n = 3). I–N: The protein levels of CXCR4, TRAP, N-cadherin, E-cadherin, and vimentin in the four groups of mice by Western blotting ( n = 3). O: The CXCR4 expression in the four groups of mice. Data are presented as mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001, and ns P > 0.05, comparisons are shown in the figure. Data were analyzed using two-way ANOVA, and differences between groups were analyzed using Student's t -test.

Article Snippet: CXCL12 enzyme-linked immunosorbent assay (ELISA) Kit (Cat. #KE10046) was from Proteintech (Wuhan, China).

Techniques: Injection, Expressing, Immunohistochemical staining, Staining, Western Blot, Standard Deviation

Journal: Journal of Biomedical Research

Article Title: DEC1 promotes breast cancer bone metastasis through transcriptional activation of CXCR4

doi: 10.7555/JBR.39.20250031

Figure Lengend Snippet: DEC1 promoted CXCL12 production from mesenchymal stromal cells in mice and MC3T3-E1 cells. A: The CXCL12 expression in the femur of wildtype (WT) and Dec1 -knockout (KO) mice by immunohistochemical staining ( n = 3 in each group). B: The relative Cxcl12 mRNA levels in bone tissues from the femur of WT and Dec1 -KO mice ( n = 5 in each group). C: The relative Cxcl12 mRNA levels in mesenchymal cells from the femur of WT and Dec1 -KO mice ( n = 3 in each group). D–F: MC3T3-E1 cells were seeded into 6-well plates and cultured overnight. Cells were divided into three groups: Vector (infected with LV-shCon and transfected with Flag-CMV2), DEC1 -OE (transfected with Flag- DEC1 ), and DEC1 -KD (infected with LV-sh DEC1 ). After 24 h of transfection or infection, the culture medium from each group was collected, centrifuged, and filtered to obtain conditioned medium (CM). Effect of CM from MC3T3-E1 cells with DEC1 -OE or KD on breast cancer cell migration by Transwell migration assays (D). The relative Cxcl12 mRNA levels in MC3T3-E1 cells (E). The CXCL12 amount in CMs from DEC1 -OE and KO in MC3T3-E1 cells by ELISA assays (F). G: Mechanism of DEC1 promoting breast cancer bone metastasis via transcriptional activation of CXCR4. Data are presented as mean ± standard deviation (all experiments were repeated at least three times). * P < 0.05, ** P < 0.01, *** P < 0.001, comparisons are shown in the figure. Data were analyzed using one-way ANOVA, and differences between groups were analyzed using Student's t -test. Abbreviation: EMT, epithelial-to-mesenchymal transition; KD, knockdown; OE, overexpression.

Article Snippet: CXCL12 enzyme-linked immunosorbent assay (ELISA) Kit (Cat. #KE10046) was from Proteintech (Wuhan, China).

Techniques: Expressing, Knock-Out, Immunohistochemical staining, Staining, Cell Culture, Plasmid Preparation, Infection, Transfection, Migration, Enzyme-linked Immunosorbent Assay, Activation Assay, Standard Deviation, Knockdown, Over Expression

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Release of the chemokine CXCL12 by endothelial cells in AAGN promoted monocyte recruitment and infiltration. A Spatial transcriptome sequencing of renal tissues revealed that CCL2, CCL21 and CXCL12 were increased in AAGN group, of which CXCL12 was the highest. B The immunohistochemical staining of CXCL12 was increased in AAGN group (AAGN = 6, CTRL = 4, 80 ×, scale bar = 25 μm). C ELISA levels of CXCL12 were elevated in MPO + AAGN plasma ( n = 15) and urine ( n = 6). D ELISA levels of CXCL12 were not significantly increased in PR3 + AVV plasma ( n = 10) and PR3 + AAGN urine ( n = 9). E CXCL12 mRNA was increased in EA.hy926 after 3 days of incubation with AAGN serum medium, but not in HRMC, HPC and HK-2 ( n = 3). F Western blotting showed that after 3 days of incubation with AAGN serum medium, CXCL12 protein level was increased in EA.hy926, while not changed in HRMC, HPC and HK-2 ( n = 3). G CD14 immunofluorescence was increased in the crescents of AAGN group (AAGN = 6, CTRL = 4, PAS 80 ×, scale bar = 25 μm, IF 600 ×, scale bar = 100 μm). H Transwell assay confirmed that EA.hy926 incubated with AAGN serum in the lower chamber could recruit CD14. + monocytes from the upper chamber to migrate to the lower chamber ( n = 3, 400 ×, scale bar = 10 μm). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Sequencing, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Incubation, Western Blot, Immunofluorescence, Transwell Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Glomerular endothelial cell apoptosis in AAGN resulted in CXCL12 release. A Immunofluorescence levels of CD31 and ERG were decreased in AAGN crescents, whereas TUNEL was increased (AAGN = 6, CTRL = 4, PAS 80 ×, scale bar = 50 μm, IF 600 ×, scale bar = 20 μm). B The mRNA and protein levels of BCL-2/BAX were decreased in endothelial cells incubated with AAGN serum ( n = 3). C The mRNA and protein levels of cleaved-Caspase-3 were decreased in endothelial cells incubated with AAGN serum ( n = 3). D The mRNA and protein levels of CD31 and ERG were decreased in endothelial cells incubated with AAGN serum ( n = 3). E CCK-8 assay showed that with the prolonged incubation time of AAGN serum, the proliferation ability of endothelial cells decreased, and the most obvious was observed at 72–84h ( n = 3). F Flow cytometry verified that the proportion of apoptotic endothelial cells was significantly increased when incubated with AAGN serum for 3 days ( n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Immunofluorescence, TUNEL Assay, Incubation, CCK-8 Assay, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: CXCL12/CXCR4 modulates macrophage efferocytosis to induce glomerular crescent formation and fibrosis via ELMO1/DOCK180/RAC1 signaling in ANCA-associated glomerulonephritis

doi: 10.1007/s00018-025-05750-5

Figure Lengend Snippet: Inhibition of CXCL12/CXCR4 alleviates AAGN progression. A Suitable concentrations for LIT927 (CXCL12 neutral ligand antagonist) and AMD3100 (CXCR4 inhibitor) were determined as 30 μM and 100 μM, respectively, using CCK-8 assays ( n = 3). B When mixed individually with AAGN serum to stimulate macrophages (Mφs), both drugs reduced MERTK, CD163, TGF-β1 and ELMO1/DOCK180/RAC1 axis components at transcriptional and protein levels ( n = 3). C Schematic of the EAV rat model establishment. Drug administration, either concurrently with modeling or from the third week post-modeling, did not significantly affect rat body weight D but alleviated hematuria E and proteinuria F , with AMD3100 showing greater efficacy. Starting treatment at the third week was as effective as concurrent administration. G LIT927 administration at the onset of modeling reduced cellular and fibrous crescent formation. When started from the third week, it alleviated fibrocellular and fibrous crescents without affecting cellular crescents. AMD3100 reduced various types of crescent formation when given at modeling onset but primarily alleviated fibrocellular and fibrous crescents when started three weeks later (80 ×, scale bar = 10 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001

Article Snippet: CXCL12 (CSB-EQ027490HU for human, CSB-E08729r for rat, Cusabio, Wuhan, China) and soluble CXCR4 (CSB- E12825 h for human, CSB-E12703r for rat, Cusabio) in serum and urine were measured with ELISA kits following the manufacturer’s instructions.

Techniques: Inhibition, CCK-8 Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PPY-Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer.

doi: 10.1002/advs.202413432

Figure Lengend Snippet: Figure 1. Screening and initial validation of cancer cell-secreted proteins capable of significantly inducing iCAF phenotype. A) The t-distributed stochastic neighbor embedding (t-SNE) plot of the 92,222 cells in the single-cell sequencing profile revealed distinct cell types observed in PDAC. B) The t-SNE plot exhibited diverse subtypes of fibroblasts observed in PDAC. C) The top 10 up-regulated and down-regulated expressed marker genes of each CAF subgroup. D) The expression levels of myCAF markers (ACTA2, COL1A1, COL11A1, MMP11), iCAF markers (CXCL12, IL-6, CCL2, CXCL2), and apCAF markers (HLA-DRA, HLA-DRB1) in different fibroblast subsets. E) Pathway activities scored by GSVA between different fibroblast subsets. F) The volcano

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL-6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL-6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Biomarker Discovery, Sequencing, Marker, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PPY-Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer.

doi: 10.1002/advs.202413432

Figure Lengend Snippet: Figure 2. PPY significantly induces the iCAF phenotype in PDAC CAFs both in vitro and in vivo. A–C) After treating CAFs derived from human PDAC tissues for 12 h, qRT-PCR analysis was performed to assess their alterations in the expression of iCAF markers (CXCL12 (A), IL-6 (B), CXCL12 (C)). D–F) qRT-PCR analysis of iCAF markers (CXCL12 (D), IL-6 (E), CXCL12 (F)) after treating the human CAFs for 24 h. G–I) qRT-PCR analysis of iCAF markers (CXCL12 (G), IL-6 (H), CXCL12 (I)) after treating the human CAFs for 36 h. J) qRT-PCR analysis of the expression levels of myCAF markers (ACTA2 and CTGF) after treating the human CAFs with PPY proteins (40ng/ml) for 24 h. K) Flow cytometry analysis was performed to evaluate the

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL-6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL-6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: In Vitro, In Vivo, Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: PPY-Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer.

doi: 10.1002/advs.202413432

Figure Lengend Snippet: Figure 7. The inhibition of EGFR expression in CAFs impeded the induction of iCAFs by PPY. A) qRT-PCR (A) and B) ELISA analyses of the expression levels of IL-6, CCL2, and CXCL12 in human EGFR-knockdown CAFs treated with PPY proteins. C) The efficiency of EGFR knockdown in KPC CAFs was examined by qRT-PCR. D,E) qRT-PCR (D) and ELISA (E) analyses of the expression levels of IL-6, CCL2, and CXCL12 in murine EGFR knockdown CAFs treated with PPY proteins. F) Flow cytometry analysis was performed to evaluate the populations of iCAFs, myCAFs, and apCAFs in murine EGFR- knockdown CAFs treated with PPY proteins. G,H) The IVIS image (G) and gross image (H) of tumors in model mice (n = 7), that was constructed

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL-6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL-6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knockdown, Flow Cytometry, Construct

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: Screening and initial validation of cancer cell‐secreted proteins capable of significantly inducing iCAF phenotype. A) The t‐distributed stochastic neighbor embedding (t‐SNE) plot of the 92,222 cells in the single‐cell sequencing profile revealed distinct cell types observed in PDAC. B) The t‐SNE plot exhibited diverse subtypes of fibroblasts observed in PDAC. C) The top 10 up‐regulated and down‐regulated expressed marker genes of each CAF subgroup. D) The expression levels of myCAF markers (ACTA2, COL1A1, COL11A1, MMP11), iCAF markers (CXCL12, IL‐6, CCL2, CXCL2), and apCAF markers (HLA‐DRA, HLA‐DRB1) in different fibroblast subsets. E) Pathway activities scored by GSVA between different fibroblast subsets. F) The volcano plot depicts the differential expression of genes encoding secreted proteins in cancer cells derived from patients with high versus low iCAF. G–I) qRT‐PCR analysis was conducted to assess alterations in the expression levels of iCAF markers (IL‐6, CXCL12, and CCL2) in CAFs isolated from KPC mice following treatment with conditioned medium (CM) containing potential candidates for 12 (G), 24 (H), and 36 h (I). J) qRT‐PCR analysis of changes in the expression levels of myCAF markers (ACTA2 and CTGF) in CAFs isolated from KPC mice after treating them with CM containing PPY for 12, 24, and 36 h. K) Flow cytometry analysis of iCAF (Ly6C+MHC‐II‐), myCAF (Ly6C+MHC‐II‐), and apCAF (Ly6C+MHC‐II‐) populations after treating CAFs with CM containing PPY for 24 h. L,M) The changes in expression of iCAF markers (IL‐6, CXCL12, and CCL2) (L) and myCAF markers (ACTA2 and CTGF) (M) in CAFs isolated from three patients with PDAC were quantified by qRT‐PCR; following a 24‐h treatment with CM containing PPY. N) After treating CAFs derived from three PDAC patients with CM containing PPY for 24 h, ELISA was performed to assess the secretion of IL‐6, CCL2, and CXCL12. Each experiment was performed three times independently, and Student's t ‐test was used to analyze the data. The results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Biomarker Discovery, Sequencing, Marker, Expressing, Quantitative Proteomics, Derivative Assay, Quantitative RT-PCR, Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: PPY significantly induces the iCAF phenotype in PDAC CAFs both in vitro and in vivo. A–C) After treating CAFs derived from human PDAC tissues for 12 h, qRT‐PCR analysis was performed to assess their alterations in the expression of iCAF markers (CXCL12 (A), IL‐6 (B), CXCL12 (C)). D–F) qRT‐PCR analysis of iCAF markers (CXCL12 (D), IL‐6 (E), CXCL12 (F)) after treating the human CAFs for 24 h. G–I) qRT‐PCR analysis of iCAF markers (CXCL12 (G), IL‐6 (H), CXCL12 (I)) after treating the human CAFs for 36 h. J) qRT‐PCR analysis of the expression levels of myCAF markers (ACTA2 and CTGF) after treating the human CAFs with PPY proteins (40ng/ml) for 24 h. K) Flow cytometry analysis was performed to evaluate the populations of iCAFs (Ly6C+MHC‐II‐), myCAFs (Ly6C+MHC‐II‐), and apCAFs (Ly6C+MHC‐II‐), after treating CAFs derived from cancer tissues of KPC mice with PPY recombinant proteins. L,M) After co‐culturing the human CAFs together with BxPC‐3 cells overexpressing PPY, the expression levels of iCAF markers (IL‐6, CCL2, and CXCL12) and myCAF markers (ACTA2 and CTGF) were quantified using qRT‐PCR (L), and the secretion levels of IL‐6, CCL2, and CXCL12 were measured using ELISA (M). N) The CAFs derived from cancer tissues of KPC mice were cocultured with Panc02 overexpressed PPY, and flow cytometry was applied to analyze iCAF, myCAF, and apCAF populations. O,P) After co‐culturing the human CAFs with PANC‐1 cells that had down‐regulated PPY expression, the expression levels of iCAF markers (IL‐6, CCL2, and CXCL12) and myCAF markers (ACTA2 and CTGF) were analyzed by qRT‐PCR (L), and secretion levels of IL‐6, CCL2, and CXCL12 were assessed by ELISA (M). Q) The murine CAFs were cocultured with Panc02 that had down‐regulated PPY expression, and flow cytometry was applied to analyze iCAF, myCAF, and apCAF populations. R) Schematic diagram of co‐injection of mouse cancer cells and CAFs (4:1) derived from KPC mice to construct the orthotopic allograft tumor model in C57BL/6J mice (n = 8), and the tumor tissues were isolated and analyzed by flow cytometry. S,T) The tumor tissues of PPY upregulated and downregulated groups and their respective control groups were dissociated into single cells, and flow cytometry was utilized to analyze the iCAF, myCAF, and apCAF populations in the tumor tissue. Student's t ‐test was used to analyze the data, and the results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ***, p < 0.001; ns, not statistically significant. OE, overexpression.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: In Vitro, In Vivo, Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Recombinant, Enzyme-linked Immunosorbent Assay, Injection, Construct, Isolation, Control, Over Expression

Journal: Advanced Science

Article Title: PPY‐Induced iCAFs Cultivate an Immunosuppressive Microenvironment in Pancreatic Cancer

doi: 10.1002/advs.202413432

Figure Lengend Snippet: The inhibition of EGFR expression in CAFs impeded the induction of iCAFs by PPY. A) qRT‐PCR (A) and B) ELISA analyses of the expression levels of IL‐6, CCL2, and CXCL12 in human EGFR‐knockdown CAFs treated with PPY proteins. C) The efficiency of EGFR knockdown in KPC CAFs was examined by qRT‐PCR. D,E) qRT‐PCR (D) and ELISA (E) analyses of the expression levels of IL‐6, CCL2, and CXCL12 in murine EGFR knockdown CAFs treated with PPY proteins. F) Flow cytometry analysis was performed to evaluate the populations of iCAFs, myCAFs, and apCAFs in murine EGFR‐knockdown CAFs treated with PPY proteins. G,H) The IVIS image (G) and gross image (H) of tumors in model mice (n = 7), that was constructed by co‐injecting cancer cells with up‐regulated PPY expression and KPC CAFs with down‐regulated EGFR expression. I–N) Flow cytometric analysis was performed to evaluate the presence of iCAFs (I), M2 macrophages (J), MDSCs (K), T cells (L), CD8 + T cells (M), and exhausted CD8 + T cells (N) within the tumor microenvironment. O) Map of scientific hypotheses of this article. The statistical data is presented as mean ± SD and analyzed using the unpaired t‐ test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: ELISA assays used were Mouse CXCL12 ELISA kit (KE10049, Proteintech), Mouse IL‐6 ELISA kit (EK206HS, MULTI SCIENCES), Mouse CCL2 ELISA kit (EK287, MULTI SCIENCES), Human CXCL12 ELISA kit (EK1119, MULTI SCIENCES), Human CCL2 ELISA kit (EK187, MULTI SCIENCES), and Human IL‐6 ELISA kit (EK106, MULTI SCIENCES).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Knockdown, Flow Cytometry, Construct