Additional file 1 of Temperature-induced embryonic diapause in chickens is mediated by PKC-NF-κB-IRF1 signaling
Creators: name: Ren, Junxiao
name: Hu, Zhengzheng
name: Li, Quanlin
name: Gu, Shuang
name: Lan, Fangren
name: Wang, Xiqiong
name: Li, Jianbo
name: Li, Junying
name: Shao, Liwa
name: Yang, Ning
name: Sun, Congjiao
Publisher: name: figshare
Year: 2023
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. Complementary data shows the differences between pre-diapause and diapause embryos. (A), Immunostaining of blastoderm sections with an antibody against the proliferation marker PCNA. Samples were counterstained with DAPI to visualize DNA (blue). Scale bar, 40 μm. (B) Differences in expression between blastoderms isolated from fresh oviposited eggs (F0) or stored at low temperature for 8 days (S8d) of genes associated with cell proliferation, N = 4. (C), TUNEL assay of blastocysts (green). Samples were counterstained with DAPI to visualize DNA (blue). Scale bar, 40 μm. (D), Box plots showing the abundance of representative metabolites in F0 or S8d blastoderm. (E), Differences in expression between blastoderms isolated from F0 and S8d of genes encoding key enzyme in the TCA cycle, N = 4. Fig. S2. Data summary of the RNA-Seq data generated during the initiation and maintaining of diapause. (A), Evaluation of RNA-Seq data generated from blastoderms isolated from fresh oviposited or low temperature-stored eggs. (B), Venn diagram showing the number of DEGs identified by RNA-Seq. (C, D), RNA-Seq expression levels of proliferation associated genes (C), and pluripotency markers genes (D), represented as heatmaps. Genes involved in negative regulation proliferation are marked in red while genes involved in negative regulation proliferation are marked in blue. Fig. S3. Data summary of the RNA-Seq data generated during the termination of diapause. (A), Evaluation of RNA-Seq data generated from blastoderms isolated from incubated eggs. (B), Scatterplot comparing the fold change of gene expression during the reactivation process. Each dot represents a gene quantified by RNA-Seq. Fig. S4. Complementary data for Fig. 4E shows the expressions and functions of the screened genes. (A), RNA-Seq data showing dynamic changes in the expression of genes at all time points. (B), DAVID analysis of enriched GO terms for the 19 screened genes. (C), Protein-protein interaction network of the screened genes. The line thickness indicates the strength of data support. (D), qPCR of changes in gene expression after ESCs were induced by cold stress (28°C) for 3 hours (C3h) or 6 hours (C6h), N = 5. Fig. S5. Data summary of the phosphoproteomic data. (A), Heatmap showing the Pearson correlation coefficients between the different biological replicates. (B), Principal component analysis of phosphoproteomic data. (C), Distribution of serine (S), threonine (T) and tyrosine (Y) phosphorylation sites. (D), Number of significantly regulated phosphopeptides identified by different comparations. The threshold was set as P value &lt; 0.05 and fold change &gt;1.5. (E), Heatmap shows the phosphorylation levels of cell cycle associated proteins. (F), Histogram of phosphorylation levels of the indicated phosphosites in Marcks and Marcksl1 peoteins. Fig. S6. mTOR pathways is not significantly changed during the transition between pre-diapause and diapause. (A), RNA-Seq expression levels of mTOR related genes. The size and color show the average RNA-Seq normalized expression values across replicates. (B), Phosphoproteomics data shows the phosphorylation level of mTOR target protein sites. Data was represented as heatmap, color show the normalized phosphoproteomics values. (C), Western blot analysis of the pre-diapause and diapause embryos with ribosome protein S6 (Cat: 2217S; CST) and its phosphorylated form pS6 (Cat: 2211S; CST) antibody. GAPDH was used as used as the loading control. Fig. S7. Changes in gene expression after ESCs were stimuli by cold stress (28°C) for 3 hours (C3h) or 6 hours (C6h), N = 5. Fig. S8. Complementary data for Fig. 7 shows effects of OE-IRF1 on cell cycle and embryonic growth. (A), Representative flow cytometry histogram showing cell cycle analysis. (B), Statistics of cell cycle results from (B) based on three technical replicates. (C), Verification of the efficacy of lentiviral mediated IRF1 overexpression (OE-IRF1) in blastoderms, N = 5. (D), Changes in diameter of the blastoderms after IRF1 overexpression, N = 6. For all figures, mean ± SEM is shown. *p &lt;0.05; **p &lt; 0.01; ***p &lt;0.001.