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pgl3basic vectors  (Promega)

 
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    Promega pgl3basic vectors
    Pgl3basic Vectors, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pgl3basic+vector/pgl3+basic/pm40253379-413-25-27
    Average 90 stars, based on 1 article reviews
    pgl3basic vectors - by Bioz Stars, 2026-10
    90/100 stars

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    Related Articles

    Sequencing:

    Article Title: TFAP2A is involved in neuropathic pain by regulating Grin1 expression in glial cells of the dorsal root ganglion.
    Article Snippet: Neuropathic pain is a highly prevalent and refractory condition, yet its mechanism remains poorly understood.. While NR1, the essential subunit of NMDA receptors, has long been recognized for its pivotal role in nociceptive transmission, its involvement in presynaptic stimulation is incompletely elucidated.. Transcription factors can regulate the expression of both pro-nociceptive and analgesic factors.

    Article Title: Pulsatile Flow Increases METTL14-induced m6A modification and attenuates septic cardiomyopathy: an experimental study.
    Article Snippet: Using the specific m6A antibody (202003, Synaptic Systems, Goettingen, Germany), the mRNAs spotted on the Hybond N+ membranes were detected by dot blot assays. .. Plasmid reconstruction and lentivirus infection The METTL14 promoter region (sequence -2000 bp to 100 bp) was constructed to the pGL3basic vector (E1751, Promega, Beijing, China), in which the reporter plasmid was established. .. To construct shRNA of Foxo1, obligos for Foxo1 mRNA (Supplementary Table 2, Supplemental Digital Content 2, http://links.lww.com/JS9/C241) were synthesized (Genepharma, Soochow, China) and cloned into the pLKO.1-puro vector (QP1765, Qiyunbio.

    Transformation Assay:

    Article Title: TFAP2A is involved in neuropathic pain by regulating Grin1 expression in glial cells of the dorsal root ganglion.
    Article Snippet: Neuropathic pain is a highly prevalent and refractory condition, yet its mechanism remains poorly understood.. While NR1, the essential subunit of NMDA receptors, has long been recognized for its pivotal role in nociceptive transmission, its involvement in presynaptic stimulation is incompletely elucidated.. Transcription factors can regulate the expression of both pro-nociceptive and analgesic factors.

    Luciferase:

    Article Title: Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma
    Article Snippet: .. Luciferase assay For assessing the promoter activity of SELENOW, the SELENOW reporter plasmid (kindly donated by Dr. Ick Young Kim, Korea University) was constructed by cloning a PCR product spanning − 973 to + 32 at the upstream position of the start codon of SELENOW into a KpnI/HindIII site of luciferase-encoding pGL3Basic vector (Promega®, Madison, WI, USA). .. The reporter plasmid was transfected into RAW264.7 cells by nucleoporation according to the manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany).

    Article Title: Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.
    Article Snippet: IC50 values were calculated using GraphPad Prism v7 software (GraphPad Prism). .. The GLI-responsive luciferase reporter plasmid (8 ×3’ Gli-BS) [41] was used in combination with Renilla luciferase pRL-TK reporter vector (Promega, Madison, WI) to normalize luciferase activities, and the pGL3Basic vector (Promega) was used to normalize DNA amounts. .. Luminescence was measured using the Dual-Glo Luciferase Assay System (Promega) and GloMax 20/20 Luminometer (Promega).

    Activity Assay:

    Article Title: Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma
    Article Snippet: .. Luciferase assay For assessing the promoter activity of SELENOW, the SELENOW reporter plasmid (kindly donated by Dr. Ick Young Kim, Korea University) was constructed by cloning a PCR product spanning − 973 to + 32 at the upstream position of the start codon of SELENOW into a KpnI/HindIII site of luciferase-encoding pGL3Basic vector (Promega®, Madison, WI, USA). .. The reporter plasmid was transfected into RAW264.7 cells by nucleoporation according to the manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany).

    Plasmid Preparation:

    Article Title: Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma
    Article Snippet: .. Luciferase assay For assessing the promoter activity of SELENOW, the SELENOW reporter plasmid (kindly donated by Dr. Ick Young Kim, Korea University) was constructed by cloning a PCR product spanning − 973 to + 32 at the upstream position of the start codon of SELENOW into a KpnI/HindIII site of luciferase-encoding pGL3Basic vector (Promega®, Madison, WI, USA). .. The reporter plasmid was transfected into RAW264.7 cells by nucleoporation according to the manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany).

    Article Title: Pulsatile Flow Increases METTL14-induced m6A modification and attenuates septic cardiomyopathy: an experimental study.
    Article Snippet: Using the specific m6A antibody (202003, Synaptic Systems, Goettingen, Germany), the mRNAs spotted on the Hybond N+ membranes were detected by dot blot assays. .. Plasmid reconstruction and lentivirus infection The METTL14 promoter region (sequence -2000 bp to 100 bp) was constructed to the pGL3basic vector (E1751, Promega, Beijing, China), in which the reporter plasmid was established. .. To construct shRNA of Foxo1, obligos for Foxo1 mRNA (Supplementary Table 2, Supplemental Digital Content 2, http://links.lww.com/JS9/C241) were synthesized (Genepharma, Soochow, China) and cloned into the pLKO.1-puro vector (QP1765, Qiyunbio.

    Article Title: Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.
    Article Snippet: .. Three deletion fragments (1599 bp, 1097 bp and 447 bp) of the promoter were amplified from genomic DNA according to the designed primers (Additional file 1: Table S1), and the PCR products were purified and cloned into the pGL3basic vector (Promega, USA) using KpnI and HindIII. .. Site-directed mutagenesis for the stat3 binding sites was performed using a Fast Site-Directed Mutagenesis Kit (Tiangen, China), and the primers are described in Additional file 1: Table S1.

    Article Title: Integrative analysis of the ST6GALNAC family identifies GATA2-upregulated ST6GALNAC5 as an adverse prognostic biomarker promoting prostate cancer cell invasion.
    Article Snippet: .. Intron 1 of ST6GALNAC5 gene containing the putative GATA2 binding motif (CGATA) was cloned into pGL3Basic vector (Promega, Madison, WI) as the ST6GALNAC5-Luc reporter, while the GATA2 binding motif was mutated to CTTTA as described previously [20], using Mut Express II Fast Mutagenesis Kit V2 (C214-02; Vazyme). ..

    Article Title: Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.
    Article Snippet: IC50 values were calculated using GraphPad Prism v7 software (GraphPad Prism). .. The GLI-responsive luciferase reporter plasmid (8 ×3’ Gli-BS) [41] was used in combination with Renilla luciferase pRL-TK reporter vector (Promega, Madison, WI) to normalize luciferase activities, and the pGL3Basic vector (Promega) was used to normalize DNA amounts. .. Luminescence was measured using the Dual-Glo Luciferase Assay System (Promega) and GloMax 20/20 Luminometer (Promega).

    Construct:

    Article Title: Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma
    Article Snippet: .. Luciferase assay For assessing the promoter activity of SELENOW, the SELENOW reporter plasmid (kindly donated by Dr. Ick Young Kim, Korea University) was constructed by cloning a PCR product spanning − 973 to + 32 at the upstream position of the start codon of SELENOW into a KpnI/HindIII site of luciferase-encoding pGL3Basic vector (Promega®, Madison, WI, USA). .. The reporter plasmid was transfected into RAW264.7 cells by nucleoporation according to the manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany).

    Article Title: Pulsatile Flow Increases METTL14-induced m6A modification and attenuates septic cardiomyopathy: an experimental study.
    Article Snippet: Using the specific m6A antibody (202003, Synaptic Systems, Goettingen, Germany), the mRNAs spotted on the Hybond N+ membranes were detected by dot blot assays. .. Plasmid reconstruction and lentivirus infection The METTL14 promoter region (sequence -2000 bp to 100 bp) was constructed to the pGL3basic vector (E1751, Promega, Beijing, China), in which the reporter plasmid was established. .. To construct shRNA of Foxo1, obligos for Foxo1 mRNA (Supplementary Table 2, Supplemental Digital Content 2, http://links.lww.com/JS9/C241) were synthesized (Genepharma, Soochow, China) and cloned into the pLKO.1-puro vector (QP1765, Qiyunbio.

    Cloning:

    Article Title: Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma
    Article Snippet: .. Luciferase assay For assessing the promoter activity of SELENOW, the SELENOW reporter plasmid (kindly donated by Dr. Ick Young Kim, Korea University) was constructed by cloning a PCR product spanning − 973 to + 32 at the upstream position of the start codon of SELENOW into a KpnI/HindIII site of luciferase-encoding pGL3Basic vector (Promega®, Madison, WI, USA). .. The reporter plasmid was transfected into RAW264.7 cells by nucleoporation according to the manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany).

    Polymerase Chain Reaction:

    Article Title: Selenoprotein W engages in overactive osteoclast differentiation in multiple myeloma
    Article Snippet: .. Luciferase assay For assessing the promoter activity of SELENOW, the SELENOW reporter plasmid (kindly donated by Dr. Ick Young Kim, Korea University) was constructed by cloning a PCR product spanning − 973 to + 32 at the upstream position of the start codon of SELENOW into a KpnI/HindIII site of luciferase-encoding pGL3Basic vector (Promega®, Madison, WI, USA). .. The reporter plasmid was transfected into RAW264.7 cells by nucleoporation according to the manufacturer’s instructions (Amaxa Biosystems, Cologne, Germany).

    Article Title: Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.
    Article Snippet: .. Three deletion fragments (1599 bp, 1097 bp and 447 bp) of the promoter were amplified from genomic DNA according to the designed primers (Additional file 1: Table S1), and the PCR products were purified and cloned into the pGL3basic vector (Promega, USA) using KpnI and HindIII. .. Site-directed mutagenesis for the stat3 binding sites was performed using a Fast Site-Directed Mutagenesis Kit (Tiangen, China), and the primers are described in Additional file 1: Table S1.

    Article Title: Direct interaction between CREB5 and OLIG2 enhances the self-renewal and proliferation of glioma stem cells
    Article Snippet: Page 10/20 Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis To identify the genes that were downregulated by shCREB5, we performed KEGG pathway analysis using the ShinyGO software (version 0.75), which can be accessed at http://bioinformatics.sdstate.edu/go/. (45) Luciferase reporter assay To amplify the OLIG2 promoter fragment, Human Genomic DNA (WI, Promega) was used as the source material. .. The OLIG2 promoter region was obtained by PCR ampli cation and then ligated into the pGL3Basic vector (WI, Promega) following the manufacturer's instructions. .. HEK293T cells were transfected in a 24-well plate at 70% con uence using Lipofectamine 2000 (CA, Thermo Scienti c).

    Infection:

    Article Title: Pulsatile Flow Increases METTL14-induced m6A modification and attenuates septic cardiomyopathy: an experimental study.
    Article Snippet: Using the specific m6A antibody (202003, Synaptic Systems, Goettingen, Germany), the mRNAs spotted on the Hybond N+ membranes were detected by dot blot assays. .. Plasmid reconstruction and lentivirus infection The METTL14 promoter region (sequence -2000 bp to 100 bp) was constructed to the pGL3basic vector (E1751, Promega, Beijing, China), in which the reporter plasmid was established. .. To construct shRNA of Foxo1, obligos for Foxo1 mRNA (Supplementary Table 2, Supplemental Digital Content 2, http://links.lww.com/JS9/C241) were synthesized (Genepharma, Soochow, China) and cloned into the pLKO.1-puro vector (QP1765, Qiyunbio.

    Amplification:

    Article Title: Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.
    Article Snippet: .. Three deletion fragments (1599 bp, 1097 bp and 447 bp) of the promoter were amplified from genomic DNA according to the designed primers (Additional file 1: Table S1), and the PCR products were purified and cloned into the pGL3basic vector (Promega, USA) using KpnI and HindIII. .. Site-directed mutagenesis for the stat3 binding sites was performed using a Fast Site-Directed Mutagenesis Kit (Tiangen, China), and the primers are described in Additional file 1: Table S1.

    Purification:

    Article Title: Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.
    Article Snippet: .. Three deletion fragments (1599 bp, 1097 bp and 447 bp) of the promoter were amplified from genomic DNA according to the designed primers (Additional file 1: Table S1), and the PCR products were purified and cloned into the pGL3basic vector (Promega, USA) using KpnI and HindIII. .. Site-directed mutagenesis for the stat3 binding sites was performed using a Fast Site-Directed Mutagenesis Kit (Tiangen, China), and the primers are described in Additional file 1: Table S1.

    Clone Assay:

    Article Title: Regulatory mechanism of LncRNAs in gonadal differentiation of hermaphroditic fish, Monopterus albus.
    Article Snippet: .. Three deletion fragments (1599 bp, 1097 bp and 447 bp) of the promoter were amplified from genomic DNA according to the designed primers (Additional file 1: Table S1), and the PCR products were purified and cloned into the pGL3basic vector (Promega, USA) using KpnI and HindIII. .. Site-directed mutagenesis for the stat3 binding sites was performed using a Fast Site-Directed Mutagenesis Kit (Tiangen, China), and the primers are described in Additional file 1: Table S1.

    Article Title: Integrative analysis of the ST6GALNAC family identifies GATA2-upregulated ST6GALNAC5 as an adverse prognostic biomarker promoting prostate cancer cell invasion.
    Article Snippet: .. Intron 1 of ST6GALNAC5 gene containing the putative GATA2 binding motif (CGATA) was cloned into pGL3Basic vector (Promega, Madison, WI) as the ST6GALNAC5-Luc reporter, while the GATA2 binding motif was mutated to CTTTA as described previously [20], using Mut Express II Fast Mutagenesis Kit V2 (C214-02; Vazyme). ..

    Binding Assay:

    Article Title: Integrative analysis of the ST6GALNAC family identifies GATA2-upregulated ST6GALNAC5 as an adverse prognostic biomarker promoting prostate cancer cell invasion.
    Article Snippet: .. Intron 1 of ST6GALNAC5 gene containing the putative GATA2 binding motif (CGATA) was cloned into pGL3Basic vector (Promega, Madison, WI) as the ST6GALNAC5-Luc reporter, while the GATA2 binding motif was mutated to CTTTA as described previously [20], using Mut Express II Fast Mutagenesis Kit V2 (C214-02; Vazyme). ..

    Mutagenesis:

    Article Title: Integrative analysis of the ST6GALNAC family identifies GATA2-upregulated ST6GALNAC5 as an adverse prognostic biomarker promoting prostate cancer cell invasion.
    Article Snippet: .. Intron 1 of ST6GALNAC5 gene containing the putative GATA2 binding motif (CGATA) was cloned into pGL3Basic vector (Promega, Madison, WI) as the ST6GALNAC5-Luc reporter, while the GATA2 binding motif was mutated to CTTTA as described previously [20], using Mut Express II Fast Mutagenesis Kit V2 (C214-02; Vazyme). ..



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    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 <t>Luciferase</t> 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 <t>Renilla</t> 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. –
    Pgl3basic Vector, supplied by Promega, 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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    dual-luciferase reporter vector pgl3basic  (Promega)
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    Promega dual-luciferase reporter vector pgl3basic
    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 <t>Luciferase</t> 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 <t>Renilla</t> 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. –
    Dual Luciferase Reporter Vector Pgl3basic, supplied by Promega, 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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    pgl3basic luciferase reporter vector  (Promega)
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    Promega pgl3basic luciferase reporter vector
    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 <t>Luciferase</t> 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 <t>Renilla</t> 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. –
    Pgl3basic Luciferase Reporter Vector, supplied by Promega, 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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    Image Search Results


    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: The luciferase reporter assay was performed by transfecting 0.2 μg of firefly and Renilla luciferase reporter vectors (Addgene, #128046) driven by either the wild-type mouse Lcn2 or Ccl2 promoter (−1999/+1) or their respective mutants (in which the NF-κB or c-Jun DNA binding site was mutated) (Azenta) into BMMSCs seeded in 96-well plates using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, #L3000001).

    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