Journal: Reproduction (Cambridge, England)

Article Title: mTORC1-dependent suppression of autophagic activity in somatic cell nuclear transfer mouse embryos

doi: 10.1530/REP-25-0338

Figure Lengend Snippet: Preimplantation autophagic activity compared in IVF and SCNT embryos. (A) Representative fluorescent images of IVF and SCNT embryos at various times after fertilization or activation. From left to right, 24, 48, 72, and 96 h later were shown, IVF embryos on the upper row, and SCNT embryos on the lower row. GFP-LC3 (green), RFP-LC3ΔG (red). (B) Preimplantation autophagic activity in IVF and SCNT embryos. The x -axis shows the elapsed time, and the y -axis shows the relative RFP/GFP values for each embryo based on the 24-h time point. IVF (blue), SCNT (orange), lighter-colored areas indicate SEM. Two-tailed t -tests were performed at 36, 48, 60, and 72 h. IVF n = 35, SCNT n = 6. (C) Autophagic activity in IVF and SCNT embryos that were treated to enhance gene expression from 24 to 72 h after insemination or activation. The x -axis shows the elapsed time, and the y -axis shows the relative RFP/GFP values for each embryo based on the 24-h time point. IVF (blue), Kdm4d-overexpressed (red), TSA-treated (green), lighter-colored areas indicate SEM. IVF: n = 12, Kdm4d: n = 38, TSA: n = 18. (D) Autophagic activity up to 48 h after activation in SCNT embryos treated to enhance gene expression. Kdm4d-overexpressed (red), TSA-treated (green), lighter-colored areas indicate SEM. The broken lines indicate embryos that were subjected to the respective treatments, but embryonic development was arrested after the 4-cell stage. Two-tailed t -tests were performed at 48 h for each treatment group. Kdm4d arrest n = 22, TSA arrest n = 35.

Article Snippet: Briefly, the pcDNA3.1 plasmid carrying the full-length mouse Kdm4d sequence (61553, Addgene, USA) was linearized by XbaI, and it was used as a template for in vitro transcription using mMESSAGE mMACHINE T7 ULTRA Transcription kits (AM1345, Thermo Fisher Scientific).

Techniques: Activity Assay, Activation Assay, Two Tailed Test, Gene Expression

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