Journal: Scientific Reports

Article Title: Regulation of autophagy and its role in late preimplantation during mouse embryo development

doi: 10.1038/s41598-025-11359-2

Figure Lengend Snippet: Rescue of autophagy inhibition by AA supplementation. Developmental rates ( a ) and developmental results ( b ) following culture with 10 μM CQ treatment from the 4/8-cell stages to the blastocyst stage with/without AA supplementation. Scale bars represent 20 μm. ( c ) The embryos at the morula stage, cultured with CQ and AA as shown in panel ‘a,’ were immunostained for Cdx2 and Nanog, as well as with DAPI. Scale bars represent 50 μm. ( d ) The graph shows the numbers of DAPI-positive cells counted in the embryos, as shown in ( c ). Tukey–Kramer’s HSD test, CZB vs. CZB + AA + CQ p = 0.007, CZB + CQ vs. CZB + AA + CQ p = 0.0015. ( e ) The graph shows the proportion of Cdx2- and Nanog-positive cells per total cell number counted in the embryos, as shown in ( c ). Tukey–Kramer’s HSD test, Cdx2: CZB + CQ vs. CZB + AA + CQ p = 0.0012. Nanog: CZB vs. CZ + AA + CQ p = 0.0418, CZB + AA vs. CZB + CQ p = 0.0286, CZB + CQ vs. CZB + AA + CQ p = 0.0005. ( f ) The proportion of positive/negative cells in each embryo, as shown in ( c ). The cells are classified into four types: Nanog-positive (Nanog + , green), Cdx2-positive (Cdx2 + , red), double positive for Nanog and Cdx2 (Nanog + /Cdx2 + , yellow), and double negative (Nanog-/Cdx2-, gray). Tukey–Kramer’s HSD test, Cdx2-/Nanog-: CZB vs. CZB + CQ p = 0.0034, CZB + CQ vs. CZB + AA + CQ p = 0.0045. Cdx2 + /Nanog + : CZB vs. CZB + CQ p = 0.0035. ( g ) The embryos at the morula stage, cultured with CQ and AA as shown in ( a ), were immunostained for TFAP2C and Nanog, as well as with DAPI. Scale bars represent 50 μm. ( h ) The graph shows the proportion of TFAP2C- and Nanog-positive cells per total cell number counted in the embryos, as shown in ( g ). Tukey–Kramer’s HSD test, TFAP2C: no significant differences were observed . Nanog: CZB + CQ vs. CZB + AA + CQ p = 0.0322. ( i ) Relative fluorescence intensity of Nanog (green) and TFAP2C (red) in embryos shown in ( g ). Calibration was performed using DAPI fluorescence intensity.

Article Snippet: The anti-CDX2 monoclonal antibody( 1:500; BioGenex, San Ramon, CA, USA, MU392A-UC) to detect TE cells, anti-Nanog rabbit polyclonal antibody (1:500; Abcam, Cambridge, UK, ab80892) to detect the ICM cells and anti-TFAP2C(1:500; AP-2γ antibody; Santa Cruz Biotechnology, TX, USA, sc-12762) were the primary antibodies used.

Techniques: Inhibition, Cell Culture, Fluorescence

Journal: Protein & Cell

Article Title: Setd2 overexpression rescues bivalent gene expression during SCNT-mediated ZGA

doi: 10.1093/procel/pwaf010

Figure Lengend Snippet: Shortening the length of H3K4me3 domains in 2-cell stage NT embryos improves cloning efficiency. (A) Heatmap displays the categories of H3K4me3 domains on promoters in nuclear transfer 2-cell stage embryos (NT 2-C), nuclear transfer 2-cell stage embryos with WDR5-0103 treatment (NT 2-C + WDR5-0103) and natural fertilized 2-cell stage embryos (NF 2-C). H3K4me3 domains classified into four groups: broad domains, medium domains, narrow domains, and control domains. Dashed lines represent classification based on NT 2-C sample. (B) Genome browser view shows H3K4me3 enrichment of NT 2-C, NT 2-C + WDR5-0103 and NF 2-C samples at a random region of chromosome 14. The dashed box displays the genome browser view of H3K4me3 enrichment on Elf5 gene locus. H3K4me3 enrichment calculated as log 2 (H3K4me3 RPKM / input RPKM). (C) Boxplot illustrates the averaged breadth of H3K4me3 domain at all RefSeq gene promoters in NT 2-C, NT 2-C + WDR5-0103, and NF 2-C samples. Data represented as the mean ± SD. Significance analyzed using Student’s t -test (*** P < 0.001). (D) Averaged expression level [log 2 (FPKM + 1)] of 2-C (left panel) and 4-C (right panel) obH3K4me3 genes and all H3K4me3 marked genes in NT 2-C, NT 2-C + WDR5-0103, NF 2-C, NT 4-C, NT 4-C + WDR5-0103 and NF 4-C samples. Significance analyzed by using Student’s t -test (ns: not significant, * P < 0.05, **** P < 0.0001). (E) Bar chart shows the percentage of embryos reaching blastocyst stage for NT (control) and NT + WDR5-0103 (WDR5-0103) samples. Data represented as mean ± SD ( n = 7). Significance analyzed using Student’s t -test (** P < 0.01). (F) Representative images of E4.5 blastocyst of NT (control) and NT + WDR5-0103 (WDR5-0103) samples. (G) Immunostaining of OCT4 and CDX2 in control NT E4 blastocysts (control) and NT E4 blastocysts with WDR5-0103 treatment at the 2-cell stage (WDR5-0103). DAPI stains for DNA (blue), OCT4 is visualized in pink and CDX2 is visualized in green. Scale bar: 50 μm. (H) Bar chart shows the percentage of implantation rate of NT (control) and NT + WDR5-0103 (WDR5-0103) samples examined by cesarean section on E19.5. Each dashed line links a parallel experiment of control and WDR5-0103 sample. Data represented as mean ± SD ( n = 7). Significance analyzed using Student’s t -test (* P < 0.05). (I) Bar chart displaying birth rates of NT embryos without WDR5-0103 treatment (control) and with WDR5-0103 treatment at 2-cell stage (WDR5-0103). Birth rates are calculated as the number of fetuses divided by the number of 2-cells transferred to recipients. Data are represented as mean ± SD ( n ≥ 3). Significance was analyzed by using Student’s t -test (ns: not significant).

Article Snippet: For immunostaining for OCT4, CDX2, and SETD2, anti-Oct4 antibody (Abcam ab181557), anti-CDX2 antibody (BioGenex MU392A-5UC), and SETD2 polyclonal antibody (ABclonal, A3194) were used in this study.

Techniques: Cloning, Control, Expressing, Immunostaining