junb ap 1 inhibitor t5224 Search Results


ap 1 inhibitor t5224  (MedChemExpress)
99
MedChemExpress ap 1 inhibitor t5224
<t>STING-TNF-α-AP-1</t> mediated cisplatin-induced cardiomyocyte injury. (A–B) HL-1 cells were incubated with CDDP (40 μM) for a time course, and then the total protein was collected. The protein levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun, and TNF-ɑ were detected by Western blot. (C) HL-1 cells were incubated with CDDP (40 μM) for 24 h, and then the total protein was collected. The protein levels of STING, TNF-ɑ, p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot. (D) HL-1 cells were incubated with CDDP (40 μM) for 24 h, and the medium supernatant and the total protein were collected. The contents of TNF-ɑ and IL-6 in the medium supernatant were detected by ELISA and calibrated with protein concentration. (E) HL-1 cells were incubated with cGAMP (1 μg/ml) for a time course, and then the total protein was collected. The protein levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot. (F–G) Representative images and quantification of TUNEL staining in HL-1 cells. HL-1 cells were pretreated with <t>T5224</t> (10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h. Cell apoptosis induced by CDDP was detected by TUNEL staining (Green: TUNEL positive cell, DAPI: nucleus; Scale Bar: 25 μm, ×400 magnification). (H) HL-1 cells were pretreated with T5224 (10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h, and the cell viability of HL-1 cells was examined by CCK8 analysis. (I) HL-1 cells were pretreated with T5224 (1, 5, 10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h, and the total protein was collected. The protein levels of BAX were detected by Western blot ( n = 3 independent experiments; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Vehicle or DMSO group; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CDDP group).
Ap 1 Inhibitor T5224, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ap 1 inhibitor t5224  (Merck & Co)
86
Merck & Co ap 1 inhibitor t5224
(a) Representative image of AP-1 member JunB in six weeks post-stroke peri-infarct tissue and in no stroke. Scale bar = 50 µm. (b) Percentage of JunB+ tdTomato cells is significantly higher in peri-infarct tissue compared to no stroke. (c) Representative images of immunocytochemical labelling of JunB in SPCs (expressing tdTomato and GFP via the endogenous Pdgfra promoter) treated with TNFα, TNFα and the AP-1 inhibitor <t>T5224</t> or vehicle. Scale bar = 50 μm. Treatment with TNFα resulted in increased, (d) SPC migration, (e) increased GFP intensity (proxy for Pdgfra promoter activity) and (f) nuclear JunB; all of which were abolished by pre-treatment with the AP-1 inhibitor T5224. Conversely, TNFα treatment did not result in a significant increase in (g) nuclear Jun, * = P > 0.05; ** = P > 0.01 (j) Representative images of uninjured carotid artery, carotid artery one week post FeCl 3 -induced injury and carotid artery three weeks post injury in Pdgfrb-CreER T2 transgenic mice that were bred with membrane-Tomato (mT) membrane-Green Fluorescent Protein (mG) transgenic mice immunolabelled with anti-PDGFRα or (k) anti-JunB. Scale bar = 50 μm. (l) The percentage of PDGFRα-expressing cells was significantly increased at seven days post carotid artery injury and (m) the percentage of JunB+ cells was significantly elevated at 7 days post injury. (b) Repeated measures one-way ANOVA with Tukey’s post hoc test. (d-i). Mixed effects two-way ANOVA with Tukey’s multiple comparisons test. (l-m) ANOVA with Geisser-greenhouse correction and Dunnett’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.
Ap 1 Inhibitor T5224, supplied by Merck & Co, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/junb+ap+1+inhibitor+t5224/1+ap+inhibitor+t5224/bio_rxiv__64898__2025__12__10__693193-227-18-21
Average 86 stars, based on 1 article reviews
ap 1 inhibitor t5224 - by Bioz Stars, 2026-10
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ap 1 inhibitor t 5224  (MedChemExpress)
97
MedChemExpress ap 1 inhibitor t 5224
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
Ap 1 Inhibitor T 5224, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/junb+ap+1+inhibitor+t5224/T-5224/pmc12559722-310-12-15
Average 97 stars, based on 1 article reviews
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recombinant proteins fluorescein 5 isothiocyanate fitc medchemexpress cas no 3326 32 7 c fos activator protein ap 1 inhibitor  (MedChemExpress)
96
MedChemExpress recombinant proteins fluorescein 5 isothiocyanate fitc medchemexpress cas no 3326 32 7 c fos activator protein ap 1 inhibitor
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
Recombinant Proteins Fluorescein 5 Isothiocyanate Fitc Medchemexpress Cas No 3326 32 7 C Fos Activator Protein Ap 1 Inhibitor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 1 article reviews
recombinant proteins fluorescein 5 isothiocyanate fitc medchemexpress cas no 3326 32 7 c fos activator protein ap 1 inhibitor - by Bioz Stars, 2026-10
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ap1 inhibitors t5224  (ApexBio)
90
ApexBio ap1 inhibitors t5224
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
Ap1 Inhibitors T5224, supplied by ApexBio, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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t-5224 (c-fos/ap-1 inhibitor)  (Toyama Chemical Co)
90
Toyama Chemical Co t-5224 (c-fos/ap-1 inhibitor)
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
T 5224 (C Fos/Ap 1 Inhibitor), supplied by Toyama Chemical Co, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
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c fos activator protein ap 1 inhibitor  (MedChemExpress)
99
MedChemExpress c fos activator protein ap 1 inhibitor
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
C Fos Activator Protein Ap 1 Inhibitor, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ap 1 inhibitor t5224  (Selleck Chemicals)
93
Selleck Chemicals ap 1 inhibitor t5224
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
Ap 1 Inhibitor T5224, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ap 1 inhibitor t5224 - by Bioz Stars, 2026-10
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ap 1 inhibitors  (Santa Cruz Biotechnology)
92
Santa Cruz Biotechnology ap 1 inhibitors
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
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ap-1 inhibitor t-5224  (Adooq Bioscience LLC)
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Adooq Bioscience LLC ap-1 inhibitor t-5224
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 <t>the</t> <t>AP-1</t> inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific <t>inhibitor</t> <t>T-5224</t> (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. –
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STING-TNF-α-AP-1 mediated cisplatin-induced cardiomyocyte injury. (A–B) HL-1 cells were incubated with CDDP (40 μM) for a time course, and then the total protein was collected. The protein levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun, and TNF-ɑ were detected by Western blot. (C) HL-1 cells were incubated with CDDP (40 μM) for 24 h, and then the total protein was collected. The protein levels of STING, TNF-ɑ, p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot. (D) HL-1 cells were incubated with CDDP (40 μM) for 24 h, and the medium supernatant and the total protein were collected. The contents of TNF-ɑ and IL-6 in the medium supernatant were detected by ELISA and calibrated with protein concentration. (E) HL-1 cells were incubated with cGAMP (1 μg/ml) for a time course, and then the total protein was collected. The protein levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot. (F–G) Representative images and quantification of TUNEL staining in HL-1 cells. HL-1 cells were pretreated with T5224 (10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h. Cell apoptosis induced by CDDP was detected by TUNEL staining (Green: TUNEL positive cell, DAPI: nucleus; Scale Bar: 25 μm, ×400 magnification). (H) HL-1 cells were pretreated with T5224 (10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h, and the cell viability of HL-1 cells was examined by CCK8 analysis. (I) HL-1 cells were pretreated with T5224 (1, 5, 10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h, and the total protein was collected. The protein levels of BAX were detected by Western blot ( n = 3 independent experiments; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Vehicle or DMSO group; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CDDP group).

Journal: Frontiers in Pharmacology

Article Title: Activation of STING Pathway Contributed to Cisplatin-Induced Cardiac Dysfunction via Promoting the Activation of TNF-α-AP-1 Signal Pathway

doi: 10.3389/fphar.2021.711238

Figure Lengend Snippet: STING-TNF-α-AP-1 mediated cisplatin-induced cardiomyocyte injury. (A–B) HL-1 cells were incubated with CDDP (40 μM) for a time course, and then the total protein was collected. The protein levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun, and TNF-ɑ were detected by Western blot. (C) HL-1 cells were incubated with CDDP (40 μM) for 24 h, and then the total protein was collected. The protein levels of STING, TNF-ɑ, p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot. (D) HL-1 cells were incubated with CDDP (40 μM) for 24 h, and the medium supernatant and the total protein were collected. The contents of TNF-ɑ and IL-6 in the medium supernatant were detected by ELISA and calibrated with protein concentration. (E) HL-1 cells were incubated with cGAMP (1 μg/ml) for a time course, and then the total protein was collected. The protein levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot. (F–G) Representative images and quantification of TUNEL staining in HL-1 cells. HL-1 cells were pretreated with T5224 (10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h. Cell apoptosis induced by CDDP was detected by TUNEL staining (Green: TUNEL positive cell, DAPI: nucleus; Scale Bar: 25 μm, ×400 magnification). (H) HL-1 cells were pretreated with T5224 (10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h, and the cell viability of HL-1 cells was examined by CCK8 analysis. (I) HL-1 cells were pretreated with T5224 (1, 5, 10 μM) for 30 min and incubated with CDDP (40 μM) for 24 h, and the total protein was collected. The protein levels of BAX were detected by Western blot ( n = 3 independent experiments; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Vehicle or DMSO group; * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CDDP group).

Article Snippet: Cisplatin and AP-1 inhibitor T5224 were purchased from MedChemExpress (Monmouth Junction, NJ, United States).

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Protein Concentration, TUNEL Assay, Staining

(a) Representative image of AP-1 member JunB in six weeks post-stroke peri-infarct tissue and in no stroke. Scale bar = 50 µm. (b) Percentage of JunB+ tdTomato cells is significantly higher in peri-infarct tissue compared to no stroke. (c) Representative images of immunocytochemical labelling of JunB in SPCs (expressing tdTomato and GFP via the endogenous Pdgfra promoter) treated with TNFα, TNFα and the AP-1 inhibitor T5224 or vehicle. Scale bar = 50 μm. Treatment with TNFα resulted in increased, (d) SPC migration, (e) increased GFP intensity (proxy for Pdgfra promoter activity) and (f) nuclear JunB; all of which were abolished by pre-treatment with the AP-1 inhibitor T5224. Conversely, TNFα treatment did not result in a significant increase in (g) nuclear Jun, * = P > 0.05; ** = P > 0.01 (j) Representative images of uninjured carotid artery, carotid artery one week post FeCl 3 -induced injury and carotid artery three weeks post injury in Pdgfrb-CreER T2 transgenic mice that were bred with membrane-Tomato (mT) membrane-Green Fluorescent Protein (mG) transgenic mice immunolabelled with anti-PDGFRα or (k) anti-JunB. Scale bar = 50 μm. (l) The percentage of PDGFRα-expressing cells was significantly increased at seven days post carotid artery injury and (m) the percentage of JunB+ cells was significantly elevated at 7 days post injury. (b) Repeated measures one-way ANOVA with Tukey’s post hoc test. (d-i). Mixed effects two-way ANOVA with Tukey’s multiple comparisons test. (l-m) ANOVA with Geisser-greenhouse correction and Dunnett’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Journal: bioRxiv

Article Title: Ischemic stroke induces persistent alteration to brain stromal progenitor cells linked to chronic vascular dysfunction

doi: 10.64898/2025.12.10.693193

Figure Lengend Snippet: (a) Representative image of AP-1 member JunB in six weeks post-stroke peri-infarct tissue and in no stroke. Scale bar = 50 µm. (b) Percentage of JunB+ tdTomato cells is significantly higher in peri-infarct tissue compared to no stroke. (c) Representative images of immunocytochemical labelling of JunB in SPCs (expressing tdTomato and GFP via the endogenous Pdgfra promoter) treated with TNFα, TNFα and the AP-1 inhibitor T5224 or vehicle. Scale bar = 50 μm. Treatment with TNFα resulted in increased, (d) SPC migration, (e) increased GFP intensity (proxy for Pdgfra promoter activity) and (f) nuclear JunB; all of which were abolished by pre-treatment with the AP-1 inhibitor T5224. Conversely, TNFα treatment did not result in a significant increase in (g) nuclear Jun, * = P > 0.05; ** = P > 0.01 (j) Representative images of uninjured carotid artery, carotid artery one week post FeCl 3 -induced injury and carotid artery three weeks post injury in Pdgfrb-CreER T2 transgenic mice that were bred with membrane-Tomato (mT) membrane-Green Fluorescent Protein (mG) transgenic mice immunolabelled with anti-PDGFRα or (k) anti-JunB. Scale bar = 50 μm. (l) The percentage of PDGFRα-expressing cells was significantly increased at seven days post carotid artery injury and (m) the percentage of JunB+ cells was significantly elevated at 7 days post injury. (b) Repeated measures one-way ANOVA with Tukey’s post hoc test. (d-i). Mixed effects two-way ANOVA with Tukey’s multiple comparisons test. (l-m) ANOVA with Geisser-greenhouse correction and Dunnett’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Article Snippet: The day after seeding, floating debris was washed off and SPCs received either a pretreatment with the selective AP-1 inhibitor T5224 (Merck, Cat#: TA9H97BAEC5E) at 20μM or DMSO for vehicle treatment.

Techniques: Expressing, Migration, Activity Assay, Transgenic Assay, Membrane

AP-1 signaling related changes in SPCs are not mediated by IL-17. (a) Representative images of immunocytochemical labelling of JunB in SPCs (expressing tdTomato and GFP via the endogenous Pdgfra promoter) treated with IL-17, IL-17 and the AP-1 inhibitor T5224 or vehicle. Treatment with IL-17 had no effect on (b) SPC migration, (c) GFP intensity (proxy for Pdgfra promoter activity) and (d) nuclear JunB, (f) nuclear cFos or (g) nuclear Nf-κB. (e) only nuclear intensity of the AP-1 member Jun was significantly reduced upon IL-17 exposure as well as after co-treatment with the AP-1 inhibitor T5224. Scale bar = 50 μm. ANOVA with Geisser-greenhouse correction and Dunnett’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Journal: bioRxiv

Article Title: Ischemic stroke induces persistent alteration to brain stromal progenitor cells linked to chronic vascular dysfunction

doi: 10.64898/2025.12.10.693193

Figure Lengend Snippet: AP-1 signaling related changes in SPCs are not mediated by IL-17. (a) Representative images of immunocytochemical labelling of JunB in SPCs (expressing tdTomato and GFP via the endogenous Pdgfra promoter) treated with IL-17, IL-17 and the AP-1 inhibitor T5224 or vehicle. Treatment with IL-17 had no effect on (b) SPC migration, (c) GFP intensity (proxy for Pdgfra promoter activity) and (d) nuclear JunB, (f) nuclear cFos or (g) nuclear Nf-κB. (e) only nuclear intensity of the AP-1 member Jun was significantly reduced upon IL-17 exposure as well as after co-treatment with the AP-1 inhibitor T5224. Scale bar = 50 μm. ANOVA with Geisser-greenhouse correction and Dunnett’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001.

Article Snippet: The day after seeding, floating debris was washed off and SPCs received either a pretreatment with the selective AP-1 inhibitor T5224 (Merck, Cat#: TA9H97BAEC5E) at 20μM or DMSO for vehicle treatment.

Techniques: Expressing, Migration, Activity Assay

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. –

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