Table 1

Primers pairs sequence used in this study"

Fig. 1

High concentrations of CAG inhibit cell proliferation in a concentration- and time-dependent manner A: PEMFs at P3, P6, and P9 were obtained through serial passaging, and the average expression of SA-β-Gal within the cells was measured by flow cytometry; B: The cytotoxicity of CAG at varying concentrations (25-200 μmol·L-1) towards PEMFs was assessed using a CCK8 assay. High concentrations (125-200 μmol·L-1) of CAG induced significant cytotoxicity in PEMFs (P<0.01), which led to the selection of experimental CAG concentrations of 25-100 μmol·L-1 for subsequent studies; C: The cell proliferation curve of PEMFs treated with different concentrations of CAG for 7 days was presented. Only 25 μmol·L-1 CAG consistently promoted PEMF proliferation, whereas medium concentrations (50-100 μmol·L-1) of CAG inhibited PEMF proliferation in a time- and concentration-dependent manner"

Fig. 2

CAG decreases SA-β-Gal expression in a concentration- and time-dependent manner P3, P6, and P9 PEMFs were treated with 25, 50, 75, and 100 μmol·L-1 CAG for 12 h and 24 h respectively. Flow cytometric histograms of SA-β-Gal activity in these PEMFs showed that CAG reduced SA-β-Gal levels in a concentration- and time-dependent manner. The bar graph represented the quantification of the average SA-β-Gal fluorescence intensity under different treatment conditions, as shown in the corresponding flow cytometry histograms. A two-way analysis of variance was performed to analyze the SA-β-Gal flow cytometry data, *. P<0.05，**. P<0.01. Data was derived from at least three independent experiments"

Fig. 3

The effect of CAG on cell senescence A: PEMFs at P3 and P6 were treated with 100 μmol·L-1 CAG for 24 hours, and PEMFs at P9 were treated for 12 hours. Representative images of SA-β-Gal staining were shown, where the blue cells indicated by red arrows were indicative of senescent cells. Scale bar: 100 μm; B: Statistical results showed the results of SA-β-GAL positive cells in every 100 cells under the microscope. C: Western blot analysis showed p53 protein expression in PEMFs at passages P3, P6, and P9; D: Quantitative PCR analysis of the relative mRNA expression levels of SASP markers in control and CAG-treated cells. Student’s t-test was used to analyze the data, *. P<0.05, **. P<0.01"

Fig. 4

Transcriptome analysis of PEMFs treated with CAG A: Genes that were upregulated and downregulated in PEMFs; B: Comparison of gene expression results between qPCR and RNA-seq for upregulated and downregulated genes in PEMFs; C: KEGG enrichment analysis results for PEMFs; D: GO enrichment analysis results for PEMFs"

Fig. 5

Expression of focal adhesion formation-related genes and signaling pathway network diagram A: Histograms of focal adhesion-related gene expression in the control and experimental groups under PEMFs; B: Focal adhesion signaling pathway network diagram, where red indicated upregulated gene or protein expression, and green indicated downregulated gene or protein expression"

Fig. 6

The effect of CAG on β-tubulin in PEMFs PEMFs at P3 and P6 were treated with 100 μmol·L-1 CAG for 24 hours, and PEMFs at P9 were treated with 100 μmol·L-1 CAG for 12 hours. Representative β-tubulin immunofluorescence microscopy images were shown, with β-tubulin (red staining) and nuclear DNA (blue staining), Scale bar: 150 μm"

Fig. 7

The effect of CAG on vimentin in PEMFs PEMFs at P3 and P6 were treated with 100 μmol·L-1 CAG for 24 hours, and PEMFs at P9 were treated with 100 μmol·L-1 CAG for 12 hours. Representative vimentin immunofluorescence microscopy images were shown, with vimentin (green staining) and nuclear DNA (blue staining), Scale bar: 150 μm"

Fig. 8

The effect of CAG on F-actin in PEMFs A: PEMFs at P3 and P6 were treated with 100 μmol·L-1 CAG for 24 hours, and PEMFs at P9 were treated with 100 μmol·L-1 CAG for 12 hours. Representative F-actin immunofluorescence microscopy images were shown, with F-actin (green staining) and nuclear DNA (blue staining). White arrows indicated stress fibers, orange arrows indicated focal adhesion points, and red arrows indicated lamellipodia, Scale bar: 150 μm; B: Quantification of cell area; C: Measurement of stress fiber length; D: Number of stress fibers per single cell (N = 10). E: Measurement of stress fiber thickness. All data were analyzed using ImageJ software for morphological measurements, and one-way ANOVA was used for statistical analysis. *. P<0.05, **. P<0.01"

Fig. 9

CAG-treated PEMFs as nuclear donor cells promote early SCNT embryo development A: P6 PEMFs were treated with 100 μmol·L-1 CAG for 24 hours and synchronized with control group cells. SCNT was performed, and cleavage-stage embryos were photographed under a microscope 28 hours later. Blastocyst images were captured on day 6. Scale bar: 100 μm; B: After harvesting blastocysts, fluorescence staining was performed using DAPI (for DNA) and CDX2 antibodies (for trophoblast marker) on the blastocysts. Representative images were captured using a fluorescence microscope, with a blank control group included. Scale bar: 80 μm; C: The data in C represent the mean ± standard deviation from three independent experiments. One-way ANOVA statistical analysis was performed, *. P<0.05, **. P<0.01"