recombinant mouse ccl2  (R&D Systems)

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Integrated analysis of chemokine/cytokine protein after 9-Gy myeloablative radiation, HSCs, and adoptive cellular therapy and in vitro migration assays and single-cell RNA sequencing analysis in KR158B glioma. (A) Cytokine and chemokine profiling of glioma protein lysates in the tumor secretome in adoptive cellular therapy–treated gliomas and untreated control. Statistical comparisons were made using the 1-way ANOVA test. (B) CCL12 protein levels in glioma lysates were quantified by ELISA. Statistical analysis was made using the unpaired Student t test. (C) Transcriptomic analysis of chemokine ligands and receptors using nanoString digital spatial profiling. Statistical analysis was performed using the 2-way ANOVA test. (D) In vitro trans-well migration assay of MDSCs migrating in response to glioma conditioned media from excised glioma tissue combined with neutralizing antibodies against CCL2 and/or CCL12. Statistical analysis was performed using the 1-way ANOVA test. (E) ELISA CCL12 protein quantification using immune cell–specific isolates from excised glioma tissue. Statistical analysis was performed using the 1-way ANOVA test. (F) Immune cell subset deconvolution from single-cell RNA sequencing analysis from excised glioma tissue. (G) mRNA expression of CCL12 overlayed on immune cell subset deconvolution using single-cell RNA sequencing analysis from excised glioma tissue. (H) In vitro trans-well migration assay of MDSCs migration in response to immune cell-specific isolates combined with either IgG or CCL12 neutralizing antibody. Statistical comparisons conducted using the 2-way ANOVA test. * P ≤ .05. ** P ≤ .01. *** P ≤ .001. **** P ≤ .0001; error bars: mean ± standard deviation. Abbreviations: ANOVA, analysis of variance; MDSCs, myeloid-derived suppressor cells.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: In Vitro, Migration, Single Cell, RNA Sequencing, Control, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Association of Ccl2 and macrophage-associated gene expression with clinical outcomes in glioblastoma patients. (A) Kaplan-Meier analysis of disease-free survival based on Ccl2 expression. (B) Kaplan-Meier analysis of overall survival of Cd14 expression. (C) Kaplan-Meier analysis of disease-free survival stratified by Fcgr3a expression. (D) Deconvolution of single-cell RNA sequencing cell subsets in the glioma microenvironment of GBM patients. (E) Single-cell RNA sequencing deconvolution of Ccl2 expression among cell clusters from GBM patients. (F) Deconvoluted quantification of Ccl2 expression from cell clusters identified from GBM patient samples. Statistical comparisons made using the log-rank test with P values presented on the survival graphs along with hazard ratios and P values for hazard ratios.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: Gene Expression, Expressing, Single Cell, RNA Sequencing

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 upregulates expression of PD-L1 and CCL2. (A) mRNA-seq analysis of ZEB2-overexpressing SW480 cells and analysis of KEGG pathways affected by ZEB2 expression. The size of each circle represents the number of genes involved in the corresponding pathway and the color scale denotes the P-value (upper). Changes in expression of cytokine-related genes in ZEB2-overexpressing SW480 cells are vs. those in control cells (lower). (B) RT-qPCR of CCL2 , CCL28 , CXCL2 , CXCL3 , CXCL6 and CXCL12 levels in ZEB2-overexpressing vs. control SW480 cells (upper) and in ZEB2-suppressed vs. control SNU-398 cells (lower; n=4). (C) RT-qPCR of CD274 mRNA levels in ZEB2-overexpressing vs. control SW480 cells (left) and in ZEB2-suppressed vs. control SNU-398 cells (right; n=4). (D) Analysis of CCL2 and PD-L1 protein levels in ZEB2-overexpressing vs. control SW480 cells (left), in ZEB2-suppressed vs. control SNU-398 cells (middle) and in ZEB2-overexpressing vs. control PC3 cells (right). Densitometric quantification of bands on the immunoblot was performed, with β-actin or GAPDH as a loading control. (E) Reporter analysis of CD274 and CCL2 promoter activity in ZEB2-overexpressing vs. control SW480 cells (upper) and in ZEB2-suppressed vs. control SNU-398 cells (lower; n=4). (F) Flow cytometry analysis of PD-L1 expression in SW480 cells transfected with ZEB2, TWIST1, or SNAIL expression vectors (n=3). Cells treated with IFN-γ or transfected with a PD-L1 expression vector were used as positive controls. Immunoblot analysis confirmed overexpression of ZEB2 (anti-myc), TWIST1 (anti-flag) and SNAIL (anti-SNAIL). (G) ELISA to measure secreted levels of CCL2 in conditioned medium from ZEB2-suppressed vs. control SNU-398 cells (n=3). (H, I) Scatter plots of ZEB2 mRNA expression vs. CD274 (H) and CCL2 (I) mRNA expression in colorectal adenocarcinoma (data from TCGA, Firehose Legacy and TCGA, Nature 2012). Correlations were statistically analyzed using the Spearman test. Spearman's correlation coefficients and equations were automatically generated using the cBioPortal webpage tool. (J) Kaplan-Meier analysis showing the relationship between overall survival of colon cancer (CPTAC-2, Prospective, Cell 2019; n=106) and pancreatic adenocarcinoma (TCGA, Firehose Legacy; n=178) patients and expression of ZEB2 and CD274 mRNA. P-values were calculated by the log-rank test. Values represent the mean ± standard deviation. * P<0.05; ** P<0.01; *** P<0.001; N.S, not significant. ZEB2, Zinc Finger E-Box Binding Homeobox 2; PD-L1, programmed cell death 1 ligand 1; CCL2, C-C motif chemokine ligand 2; KEGG, Kyoto Encyclopedia of Genes and Genomes; RT-qPCR, reverse transcription-quantitative PCR; ELISA, enzyme-linked immunosorbent assay; TCGA, The Cancer Genome Atlas; sh, short hairpin.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot, Activity Assay, Flow Cytometry, Transfection, Plasmid Preparation, Over Expression, Enzyme-linked Immunosorbent Assay, Generated, Standard Deviation, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 cooperates with SP1 to promote transcription of CD274 and CCL2 by binding directly to their promoters. (A) SW480 cells were co-transfected with siRNA specific for SP1 (siSP1) and with a ZEB2 expression vector, for 48 h prior to immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed, with GAPDH as a loading control. (B) RT-qPCR of CD274 (upper) and CCL2 (lower) levels in SW480 cells co-transfected with siSP1 and the ZEB2 expression vector (n=4). (C) Mutation analysis of the SP1 site in the CD274 and CCL2 promoters. SW480 cells were transfected with reporter constructs containing SP1 site mutations and reporter activity was measured (n=4). Values represent mean ± SD. *** P<0.001, vs. vector + control siRNA; $$$ P<0.001, vs. ZEB2 + control siRNA. (D) ChIP analysis of the interaction between ZEB2 and SP1 and the CD274 and CCL2 promoters. Chromatin fragments from SNU-398 cells were immunoprecipitated by normal mouse IgG (lane 1), anti-ZEB2 (lane 2), or anti-SP1 (lane 3) and data were analyzed by semiquantitative PCR using CD274 (-181/-41) and CCL2 (-115/+25) promoter primers. The input control (1%) is shown in lane 4. Irrelevant regions (-807/-660 for CD274 and -1820/-1675 for CCL2 ) were also analyzed. ZEB2, Zinc Finger E-Box Binding Homeobox 2; si, small interfering; RT-qPCR, reverse transcription-quantitative PCR; PD-L1, programmed cell death 1 ligand 1; CCL2, C-C motif chemokine ligand 2; ChIP, chromatin immunoprecipitation.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Binding Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Quantitative RT-PCR, Mutagenesis, Construct, Activity Assay, Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 promotes macrophage migration and polarization in a CCL2-dependent manner. (A) Migration of THP-1-derived macrophages by conditioned medium from and co-culturing with stable SNU-398 cells (control vs. ZEB2-suppressed cells) (n=4). CCL2 was used as a positive control. * P<0.05; ** P<0.01. (B) Migration of THP-1-derived macrophages toward conditioned medium from stable SNU-398 cells in the presence of an anti-CCL2 antibody (n=4) * P<0.05; ** P<0.01 vs. no antibody + no conditioned medium; $ P<0.05; $$ P<0.01 vs. no antibody + shSCR conditioned medium. (C) RT-qPCR of M1 ( TNF , CXCL8/IL8 , IL12B , NOS2/INOS , IL1B and IL6 ) and M2 ( TGFB1 , IL10 , MRC1 , CLEC10A , CCL17 and ARG1 ) markers secreted by THP-1-derived macrophages incubated with conditioned medium from stable SNU-398 cells in the absence and presence of an anti-CCL2 antibody (n=4). * P<0.05; ** P<0.01; *** P<0.001, vs. no antibody + no conditioned medium; $ P<0.05; $$$ P<0.001, vs. no antibody + shSCR conditioned medium. Values represent mean ± SD. ZEB2, Zinc Finger E-Box Binding Homeobox 2; CCL2, C-C motif chemokine ligand 2; sh, short hairpin.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Migration, Derivative Assay, Control, Positive Control, Quantitative RT-PCR, Incubation, Binding Assay

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: ZEB2 SUMOylation through PC2 is required for ZEB2 acting as a transcriptional activator and playing subsequent cellular functions. (A) SW480 cells were transfected with ZEB2WT and ZEB2_K391/866R for 48 h prior to lysis and immunoblot analysis. (B) Reporter assay of ITGA5 (integrin α5), VIM (vimentin), VEGFA , CDH1 , CD274 and CCL2 promoter activity in SW480 cells transfected with ZEB2WT and ZEB2_K391/866R (n=4). (C) Invasion (representative fields at magnification, ×100), (D) survival and (E) anchorage-independent growth of SW480 cells transfected with ZEB2WT and ZEB2_K391/866R (n=3). (F) SW480 cells were co-transfected with shRNA specific for CBX4 (shPC2) and with a ZEB2-expression vector, for 48 h prior to lysis and immunoblot analysis. Densitometric quantification of bands on the immunoblot was performed, with GAPDH as a loading control. Values represent mean ± SD. * P<0.05; ** P<0.01; *** P<0.001; N.S, not significant. ZEB2, Zinc Finger E-Box Binding Homeobox 2; SUMO, small ubiquitin-like modifier; CCL2, C-C motif chemokine ligand 2; PD-L1, programmed cell death 1 ligand 1; VEGF, vascular endothelial growth factor; sh, short hairpin; WT, wild type; Mut, mutant.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Transfection, Lysis, Western Blot, Reporter Assay, Activity Assay, shRNA, Expressing, Plasmid Preparation, Control, Binding Assay, Ubiquitin Proteomics, Mutagenesis

Journal: International Journal of Oncology

Article Title: Cooperation between ZEB2 and SP1 upregulates PD-L1 and CCL2 to promote the immunosuppressive activity of tumor cells

doi: 10.3892/ijo.2025.5801

Figure Lengend Snippet: SUMOylation of ZEB2 is required for cooperation between ZEB2 and SP1. Reporter assay to determine transcriptional activity of SP1 in SW480 cells (n=4). (A) Cells were transfected with ZEB2WT and ZEB2_K391/866R expression vectors for 48 h. (B) Cells were co-transfected with a ZEB2 expression vector and siRNA specific for CBX4 (siPC2) for 48 h. Values represent mean ± SD. * P<0.05; ** P<0.01; *** P<0.001. (C) SW480 cells transfected with ZEB2WT and ZEB2_K391/866R expression vectors were treated with cycloheximide for the indicated times prior to lysis and immunoblot analysis. (D) A cytosolic fraction and a nuclear fraction were prepared from 293E cells transfected for 48 h with ZEB2WT and ZEB2_K391/866R expression vectors. GAPDH and PARP were used as internal controls for the cytosolic and nuclear fractions, respectively. (E) Co-immunoprecipitation analysis of the interaction between ZEB2 and SP1 in 293E cells co-transfected with ZEB2 (WT vs. K391/866R) and SP1 expression vectors. (F) Kaplan-Meier analysis showing the probability of progression-free survival of patients with colorectal adenocarcinoma (TCGA, PanCancer Atlas; n=588) in relation to CBX4 mRNA expression. (G) Overall survival of patients with colorectal adenocarcinoma (TCGA, PanCancer Atlas; n=568) in relation to expression of ZEB2 and CBX4 mRNA. P-values were calculated using the log-rank test. SUMO, small ubiquitin-like modifier; ZEB2, Zinc Finger E-Box Binding Homeobox 2; CCL2, C-C motif chemokine ligand 2; PD-L1, programmed cell death 1 ligand 1; VEGF, vascular endothelial growth factor; TCGA, The Cancer Genome Atlas; si, small interfering; WT, wild type; Mut, mutant.

Article Snippet: The amount of CCL2 protein in conditioned medium from cells was measured using a Human CCL2 Quantikine ELISA kit (cat. no. DCP00; R&D Systems).

Techniques: Reporter Assay, Activity Assay, Transfection, Expressing, Plasmid Preparation, Lysis, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Mutagenesis

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Piezo1 modulates the lineage commitment of BMMSCs in an autocrine manner. BMMSCs were isolated from femurs and tibias of 10-week-old male C57BL/6J mice and then cultured in medium containing half of the conditioned medium (CM) collected from 10-week-old male WT or KO BMMSCs and half of the osteogenic or adipogenic induction medium. a Schematic diagram showing the experimental design created with BioRender ( https://BioRender.com ). b qPCR analysis of osteogenic genes ( Runx2, Bmp2, Bglap , and Alpl ). c Representative images of alkaline phosphatase (ALP) staining. The right panel shows the quantification of the ALP-stained area via ImageJ software. d qPCR analysis of adipogenic genes ( Pparγ, C/ebpα, aP2 , and Adipoq ). e Representative images of Oil Red O staining. The right panel shows the quantitative analysis of Oil Red O staining by determining the OD520 absorbance values. Scale bar, 100 μm. f Conditioned media collected from WT and KO BMMSCs were subjected to analyses of secretory factors via an Olink Mouse Exploratory Panel, multiplex assay (Mouse Luminex® Discovery Assay, R&D Systems), and ELISA. Volcano plot showing the log2 fold change (FC) and p value (pVal) of differentially secreted proteins between Piezo1 KO and WT BMMSCs among the 97 soluble factors analyzed (log2(FC) > 1, −log10(p)<0.05). g The Nightingale rose chart displays the ranking of fold changes in the expression of 17 differentially secreted proteins between Piezo1 KO and WT BMMSCs. h Relative protein levels in the conditioned medium of WT and KO BMMSCs are presented as the fold change. i qPCR analysis of Lcn2 , Ccl2 , Prdx5 , Fst , and Matn2 in BMMSCs. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Fig. and Table

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: Isolation, Cell Culture, Staining, Software, Multiplex Assay, Luminex, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Genetic inhibition or antibody neutralization of Lcn2 could reverse the reduced osteogenesis and enhanced adipogenesis caused by Piezo1 invalidation in BMMSCs. a , b BMMSCs isolated from femurs and tibias of 10-week-old male PDGFRα-Piezo1 KO mice and their WT littermates were infected with lentivirus (multiplicity of infection [MOI] = 40) encoding eGFP together with scrambled shRNA (Lenti-sc) or shRNA against Lcn2 (Lenti-shLcn2) for 72 h in the presence of 5 μg/mL polybrene before differentiation. Osteogenic ( a ) and adipogenic ( b ) gene expression was determined by qPCR after 21 days of osteogenic differentiation and 8 days of adipogenic differentiation, respectively. c , d 200 ng/mL rabbit anti-mouse Lcn2 neutralizing antibody or non-immune rabbit IgG was added when the differentiation of BMMSCs started. qPCR analysis of osteogenic ( c ) and adipogenic genes ( d ). e – k Eight-week-old WT and KO mice were intraperitoneally injected with non-immune rabbit IgG or Lcn2 neutralizing antibody (4 mg/kg body weight) on a weekly basis for 7 weeks. The mice were then sacrificed, and their tibias were collected for further analyses. e Schematic diagram showing the experimental design created with BioRender ( https://BioRender.com ). f Representative micro-CT images showing three-dimensional trabecular bone at the proximal tibia. Scale bar, 500 μm. g Micro-CT measurements of the bone volume fraction (BV/TV), trabecular number (Tb. N), and trabecular separation (Tb. Sp) at the proximal tibia. h Representative images showing osmium-stained decalcified tibias scanned via micro-CT. Scale bar, 1000 μm. i Quantification of bone marrow adipocytes in the tibia normalized to marrow volume. j , k Circulating levels of Lcn2 ( j ) and Ccl2 ( k ) determined by ELISA. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Figs. – and Table

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: Inhibition, Neutralization, Isolation, Infection, shRNA, Gene Expression, Injection, Micro-CT, Staining, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Piezo1 suppresses Lcn2 expression and secretion via the inhibition of Ccl2-evoked NF-κB activation. BMMSCs were isolated from femurs and tibias of 10-week-old male PDGFRα-Piezo1 KO mice and WT controls. a Gene expression of Ccr2 in BMMSCs, as determined by real-time PCR. b–g Recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) was added to the adipogenic and osteogenic induction medium during BMMSC differentiation. The expression levels of osteogenic genes ( b–d ) and adipogenic genes ( e–g ) were determined via real-time qPCR. h–n WT and KO BMMSCs were treated with rmCcl2 (100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) for 24 h. h Sonicated BMMSCs were subjected to chromatin immunoprecipitation (ChIP) using an anti-p65 antibody. The ChIP DNA samples were then subjected to qPCR amplification using specific primers against the promoter region of Lcn2, as shown in supplementary Fig. . Representative gel images of end-point ChIP-qPCR products are shown. i Quantitative results are shown as the percentage of input DNA: % of Input = 2^(CT Input −CT IP )/dilution factor × 100%. j Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Lcn2 promoter (Lcn2-WT) or the mutated Lcn2 promoter with mutations in the NF-κB binding motif (Lcn2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. k Schematic diagram illustrating the actions of rmCcl2, the CCR2 antagonist, and the NF-κB-specific inhibitor QNZ on NF-κB translocation and Lcn2 expression; created with BioRender ( https://BioRender.com ). l ELISA analysis of total p65 in the nucleus and cytoplasm of BMMSCs. m Representative immunofluorescence images of intracellular p65 localization. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. n P65 nuclear translocation was quantified by ImageJ software via Pearson’s correlation coefficient (PCC) between the p65 immunofluorescence signal and DAPI staining across segmented nuclear regions. o , p WT and KO BMMSCs were treated with the NF-κB-specific inhibitor QNZ (10 nM) for another 2 h after 24-h treatment of rmCcl2 (100 ng/mL) and INCB3344 (10 nM). o The mRNA level of Lcn2 . p Lcn2 levels in the culture medium were determined by ELISA. q Illustration of how the AP-1 inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific inhibitor T-5224 (10 μM) for 24 h. r Sonicated BMMSCs were subjected to ChIP assay using an anti-c-Jun antibody. Representative gel images of end-point ChIP-qPCR products are shown. s Quantitative results are shown as the percentage of input DNA. t The mRNA level of Ccl2 . u Ccl2 levels in the culture medium were determined by ELISA. v Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Ccl2 promoter (Ccl2-WT) or the mutated Ccl2 promoter with mutations in the c-Jun binding motif (Ccl2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Figs. –

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: Expressing, Inhibition, Activation Assay, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Sonication, Chromatin Immunoprecipitation, Amplification, ChIP-qPCR, Luciferase, Reporter Assay, Transfection, Construct, Binding Assay, Sequencing, Activity Assay, Translocation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: In vivo validation of the Piezo1-Klf2-Ccl2-Lcn2 signaling axis in the regulation of osteogenesis and adipogenesis in BMMSCs. Eight-week-old male PDGFRα-Piezo1 KO mice and their WT littermates were subjected to bilateral tibial injection of lentivirus encoding eGFP together with scrambled shRNA (WT-Control or KO-Control), shRNA against Ccl2 (KO-shCcl2) or Lcn2 (KO-shLcn2), or overexpression of the mouse Klf2 gene (KO-oe Klf2) driven by the PDGFRα promoter at a dose of 1.25 × 10 6 TU per side for 7 weeks. a Representative micro-CT images showing three-dimensional trabecular bone at the proximal tibia. Scale bar, 500 μm. b Micro-CT analysis of the bone volume fraction (BV/TV), trabecular number (Tb. N.), trabecular separation (Tb. Sp.), and bone mineral density (BMD) at the proximal tibia. c , d Representative images of immunohistochemical staining of ALP in paraffin-embedded decalcified tibias ( c ) and quantification of osteoblast surface as a percentage of bone surface (Ob.S/BS, %) ( d ). Scale bar, 25 μm. e Representative images showing H&E staining of paraffin-embedded decalcified tibias. Scale bar, 50 μm. f , g Quantification of the BMA number per field ( f ) and average BMA size (μm 2 ) ( g ). h A graphical diagram summarizing the key findings in this study created with BioRender ( https://BioRender.com ). n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Fig. and Table

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: In Vivo, Biomarker Discovery, Injection, shRNA, Control, Over Expression, Micro-CT, Immunohistochemical staining, Staining