treat plasmid 54  (Addgene inc)

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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: A CRISPR-Cas9 knockout screening identifies IRF2 as a key driver of OAS3/RNase L-mediated RNA decay during viral infection

doi: 10.1073/pnas.2412725121

Figure Lengend Snippet: IRF2 promotes OAS3 expression in unstressed cells. ( A and B ) The levels of OAS3, IRF2, RNase L, and GAPDH were analyzed in the indicated U2OS ( A ) or A549 ( B ) cell lines by western blot. ( C ) The OAS3 mRNA levels were monitored by RT-qPCR in indicated U2OS ( Left ) or A549 ( Right ) cell lines. Mean values ± SD (n = 3). **** P < 0.0001 (two-tailed t test). ( D ) The levels of OAS3 were analyzed by western blot using indicated antibodies in A549 IRF2 KO cells expressing either wild-type IRF2 or DNA binding mutant IRF2K78R. ( E and F ) Total RNAs were isolated from A549 WT or IRF2 KO cells expressing wild-type IRF2, IRF2K78R, or OAS3 and monitored for integrity by a bioanalyzer after transfection with poly(I:C) (10 ng/mL, 4 h). The red arrows indicated ribosomal RNA cleavage products. ( G ) IgG and IRF2 ChIP-sequencing in A549 cells. Analysis of ChIP-sequencing data focuses on the promoter region of OAS3. (H) IRF2 ChIP was performed in A549 WT and IRF2 KO cells. IRF2 binding on the OAS3 promoter was determined by qPCR. Mean values ± SD. *** P < 0.001 (two-tailed t test). ( I ) Schematic of pGL3-OAS3-luciferase constructs harboring OAS3 WT promotor region (−1 to −900 bp upstream of transcription start site [TSS]) and IRF2 binding motif region deleted (Δ−1 to −200 bp). ( J ) The relative luciferase activity (ratio of Firefly:Renilla) was measured in U2OS cells transiently transfected with the empty pGL3 and pGL3 vector harboring either WT and Δ200 bp OAS3 promoter region. ( K ) The relative luciferase activity was monitored U2OS WT or IRF2 KO cells 24 h following transfection with the pGL3-OAS3 vector.

Article Snippet: The OAS3 luciferase reporter plasmid was generated by cloning the OAS3 promoter region (TSS −1 to −900) into pGL3 basic luciferase reporter (Addgene #128046).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Two Tailed Test, Binding Assay, Mutagenesis, Isolation, Transfection, ChIP-sequencing, Luciferase, Construct, Activity Assay, Plasmid Preparation