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pgl3 control vector  (Addgene inc)


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    Addgene inc pgl3 control vector
    Pgl3 Control Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pgl3basic+vector/pGL3+Basic+Control+Vector+(Plasmid+%23137707)/pmc12172475-208-7-30
    Average 93 stars, based on 51 article reviews
    pgl3 control vector - by Bioz Stars, 2026-10
    93/100 stars

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    Related Articles

    Binding Assay:

    Article Title: Bmal1 Regulates Vascular Calcification via Non-Canonical Circadian Pathway
    Article Snippet: The aortic expression of Runx2 and SMA in arteries from the STZ-treated Bmal1 flox/flox and Bmal1 Δ/Δ mice were determined by immunofluorescent staining of consecutive 7-μm aortic sections using specific primary antibodies (Runx2, MBL Cat# D130–3 and SMA, Sigma Cat# A5228). .. To assess a direct regulation of Bmal1 on Runx2 transcription, a 1,974-bp mouse Runx2 promoter (mm10: chr17:44,735,972–44,737,945) containing putative Bmal1 binding sites was insert into the pGL3-Basic vector (Addgene) to generate the wildtype mRunx2 promoter reporter construct (Bmal1 BS Mut −). .. Using this construct, the CACGTG Bmal1 binding site (chr17:44,737,386–44,737,391) within the mRunx2 promoter was deleted using the QuikChange Lightning Multi-Site-Directed Mutagenesis Kit (Agilent Technologies) to generate mutant mRunx2 promoter reporter construct (Bmal1 BS Mut +).

    Plasmid Preparation:

    Article Title: Bmal1 Regulates Vascular Calcification via Non-Canonical Circadian Pathway
    Article Snippet: The aortic expression of Runx2 and SMA in arteries from the STZ-treated Bmal1 flox/flox and Bmal1 Δ/Δ mice were determined by immunofluorescent staining of consecutive 7-μm aortic sections using specific primary antibodies (Runx2, MBL Cat# D130–3 and SMA, Sigma Cat# A5228). .. To assess a direct regulation of Bmal1 on Runx2 transcription, a 1,974-bp mouse Runx2 promoter (mm10: chr17:44,735,972–44,737,945) containing putative Bmal1 binding sites was insert into the pGL3-Basic vector (Addgene) to generate the wildtype mRunx2 promoter reporter construct (Bmal1 BS Mut −). .. Using this construct, the CACGTG Bmal1 binding site (chr17:44,737,386–44,737,391) within the mRunx2 promoter was deleted using the QuikChange Lightning Multi-Site-Directed Mutagenesis Kit (Agilent Technologies) to generate mutant mRunx2 promoter reporter construct (Bmal1 BS Mut +).

    Article Title: The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14
    Article Snippet: .. The BACs encoding the replicative cDNA and pGL3-control vector were transfected into HEK293T cells using TransIT-LT1 (TaKaRa) according to the manufacturer’s instructions. pGL3 Control Vector, a gift from Debrya Groskreutz (Addgene plasmid #212937; http://n2t.net/addgene:212937 ; RRID: Addgene_212937), expressed firefly luciferase and served as an internal control. .. After incubation for 72 h at 37°C, the transfected cells were lysed with Passive Lysis buffer (Promega).

    Article Title: Quercetin suppresses endometrial cancer stem cells via ERα-mediated inhibition of STAT3 signaling
    Article Snippet: .. pGL3-human Oct4 PE-SV40-luc was purchase from Addgene; the vector pGL3 bought from Pomega was used as a control. .. The protein concentrations were ascertained through the Bradford protein assay (Bio-Rad Laboratories, Milano, Italy).

    Article Title: WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression
    Article Snippet: .. To assess Wnt/β-catenin signaling activity, cells were co-transfected with either WNT3 or control Vector plasmids, along with the TopFlash/FOPFlash plasmid and the Renilla luciferase internal control plasmid (Addgene, USA). ..

    Article Title: Supporting Information for Extrachromosomal Telomere DNA Derived from Excessive Strand Displacements
    Article Snippet: The siRNAs were transfected into the cells at a final concentration of 20 nM, along with 2 l of Lipofectamine 3000 (Thermo scientific, L3000001) according to the manufacturer's protocol. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells.

    Construct:

    Article Title: Bmal1 Regulates Vascular Calcification via Non-Canonical Circadian Pathway
    Article Snippet: The aortic expression of Runx2 and SMA in arteries from the STZ-treated Bmal1 flox/flox and Bmal1 Δ/Δ mice were determined by immunofluorescent staining of consecutive 7-μm aortic sections using specific primary antibodies (Runx2, MBL Cat# D130–3 and SMA, Sigma Cat# A5228). .. To assess a direct regulation of Bmal1 on Runx2 transcription, a 1,974-bp mouse Runx2 promoter (mm10: chr17:44,735,972–44,737,945) containing putative Bmal1 binding sites was insert into the pGL3-Basic vector (Addgene) to generate the wildtype mRunx2 promoter reporter construct (Bmal1 BS Mut −). .. Using this construct, the CACGTG Bmal1 binding site (chr17:44,737,386–44,737,391) within the mRunx2 promoter was deleted using the QuikChange Lightning Multi-Site-Directed Mutagenesis Kit (Agilent Technologies) to generate mutant mRunx2 promoter reporter construct (Bmal1 BS Mut +).

    Article Title: β-catenin: A crucial transcriptional activator of KSHV latency genes and small molecule target in primary effusion lymphoma.
    Article Snippet: Latency-associated nuclear antigen (LANA)-induced β-catenin accumulation is one of the prime events in the Kaposi sarcoma-associated herpesvirus (KSHV)-associated primary effusion lymphoma (PEL).. LANA interacts with GSK3β, which in turn increases β-catenin pool in this lymphoma.. The intrinsic molecular mechanism and the effects of β-catenin accumulation in this lymphoma is still unclear. β-catenin acts as a major transcription factor that regulates a variety of proliferative signaling pathways and promotes oncogenesis.

    other:

    Article Title: Targeted protein O-GlcNAc reveals transcriptional functions for O-GlcNAc.
    Article Snippet: Technology Targeted protein O-GlcNAc reveals transcriptional functions for O-GlcNAc

    Article Title: E2F1 suppresses Epstein-Barr virus lytic reactivation through cellular and viral transcriptional networks
    Article Snippet: pGL3 Basic Vector , Gift from Debrya Groskreutz , RRID: Addgene_212936.

    Transfection:

    Article Title: The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14
    Article Snippet: .. The BACs encoding the replicative cDNA and pGL3-control vector were transfected into HEK293T cells using TransIT-LT1 (TaKaRa) according to the manufacturer’s instructions. pGL3 Control Vector, a gift from Debrya Groskreutz (Addgene plasmid #212937; http://n2t.net/addgene:212937 ; RRID: Addgene_212937), expressed firefly luciferase and served as an internal control. .. After incubation for 72 h at 37°C, the transfected cells were lysed with Passive Lysis buffer (Promega).

    Control:

    Article Title: The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14
    Article Snippet: .. The BACs encoding the replicative cDNA and pGL3-control vector were transfected into HEK293T cells using TransIT-LT1 (TaKaRa) according to the manufacturer’s instructions. pGL3 Control Vector, a gift from Debrya Groskreutz (Addgene plasmid #212937; http://n2t.net/addgene:212937 ; RRID: Addgene_212937), expressed firefly luciferase and served as an internal control. .. After incubation for 72 h at 37°C, the transfected cells were lysed with Passive Lysis buffer (Promega).

    Article Title: β-catenin: A crucial transcriptional activator of KSHV latency genes and small molecule target in primary effusion lymphoma.
    Article Snippet: Latency-associated nuclear antigen (LANA)-induced β-catenin accumulation is one of the prime events in the Kaposi sarcoma-associated herpesvirus (KSHV)-associated primary effusion lymphoma (PEL).. LANA interacts with GSK3β, which in turn increases β-catenin pool in this lymphoma.. The intrinsic molecular mechanism and the effects of β-catenin accumulation in this lymphoma is still unclear. β-catenin acts as a major transcription factor that regulates a variety of proliferative signaling pathways and promotes oncogenesis.

    Article Title: Quercetin suppresses endometrial cancer stem cells via ERα-mediated inhibition of STAT3 signaling
    Article Snippet: .. pGL3-human Oct4 PE-SV40-luc was purchase from Addgene; the vector pGL3 bought from Pomega was used as a control. .. The protein concentrations were ascertained through the Bradford protein assay (Bio-Rad Laboratories, Milano, Italy).

    Article Title: WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression
    Article Snippet: .. To assess Wnt/β-catenin signaling activity, cells were co-transfected with either WNT3 or control Vector plasmids, along with the TopFlash/FOPFlash plasmid and the Renilla luciferase internal control plasmid (Addgene, USA). ..

    Article Title: Supporting Information for Extrachromosomal Telomere DNA Derived from Excessive Strand Displacements
    Article Snippet: The siRNAs were transfected into the cells at a final concentration of 20 nM, along with 2 l of Lipofectamine 3000 (Thermo scientific, L3000001) according to the manufacturer's protocol. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells.

    Luciferase:

    Article Title: The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14
    Article Snippet: .. The BACs encoding the replicative cDNA and pGL3-control vector were transfected into HEK293T cells using TransIT-LT1 (TaKaRa) according to the manufacturer’s instructions. pGL3 Control Vector, a gift from Debrya Groskreutz (Addgene plasmid #212937; http://n2t.net/addgene:212937 ; RRID: Addgene_212937), expressed firefly luciferase and served as an internal control. .. After incubation for 72 h at 37°C, the transfected cells were lysed with Passive Lysis buffer (Promega).

    Article Title: WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression
    Article Snippet: .. To assess Wnt/β-catenin signaling activity, cells were co-transfected with either WNT3 or control Vector plasmids, along with the TopFlash/FOPFlash plasmid and the Renilla luciferase internal control plasmid (Addgene, USA). ..

    Activity Assay:

    Article Title: WNT3 promotes chemoresistance to oxaliplatin in oral squamous cell carcinoma via regulating ABCG2 expression
    Article Snippet: .. To assess Wnt/β-catenin signaling activity, cells were co-transfected with either WNT3 or control Vector plasmids, along with the TopFlash/FOPFlash plasmid and the Renilla luciferase internal control plasmid (Addgene, USA). ..

    Generated:

    Article Title: Supporting Information for Extrachromosomal Telomere DNA Derived from Excessive Strand Displacements
    Article Snippet: The siRNAs were transfected into the cells at a final concentration of 20 nM, along with 2 l of Lipofectamine 3000 (Thermo scientific, L3000001) according to the manufacturer's protocol. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells.

    shRNA:

    Article Title: Supporting Information for Extrachromosomal Telomere DNA Derived from Excessive Strand Displacements
    Article Snippet: The siRNAs were transfected into the cells at a final concentration of 20 nM, along with 2 l of Lipofectamine 3000 (Thermo scientific, L3000001) according to the manufacturer's protocol. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells. .. Lentiviruses were generated by transfecting shRNA vectors targeting human POLD3 (6), STN1 (7), ATRX(8), or the control vector, or sgRNA vectors targeting human PML exon 3 (sense: CACCGGcggtaccagcgcgactacg, antisense: AAACcgtagtcgcgctggtaccgCC) inserted in pLentiV2 vector (Addgene, 52961) along with pMD2.G and psPAX into 293T cells.



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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. –
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    90
    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
    https://www.bioz.com/product/pgl3basic+vector/pgl3+basic/pm38763227-52-13-17
    Average 90 stars, based on 1 article reviews
    dual-luciferase reporter vector pgl3basic - by Bioz Stars, 2026-10
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    pgl3basic luciferase reporter vector  (Promega)
    90
    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
    https://www.bioz.com/product/pgl3basic+vector/pgl3+basic/pm37957720-136-7-11
    Average 90 stars, based on 1 article reviews
    pgl3basic luciferase reporter vector - by Bioz Stars, 2026-10
    90/100 stars
    		  Buy from Supplier

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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 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