Additional file 1 of Transcription factor E4F1 dictates spermatogonial stem cell fate decisions by regulating mitochondrial functions and cell cycle progression
Creators: name: Yan, Rong-Ge
name: He, Zhen
name: Wang, Fei-Chen
name: Li, Shuang
name: Shang, Qin-Bang
name: Yang, Qi-En
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1 Expression and function of E4F1 during prespermatogonia to spermatogonial transition. (A) Immumohistochemical staining for E4F1 in sections of testes from PD0 and PD6 mice. n=4. The black arrows indicate E4F1 negative cells and the red arrows indicate E4F1 positive cells. (B) The Percentage of E4F1+ Cells in Germ Cells in Figure (A). (C) The expression of E4f1 mRNA from controls and E4f1 cKO testes. (D) Immunofluorescence staining for TRA98 (green) in sections of 2MO E4f1 cKO testes(panorama). n=2. (E) Immunofluorescence staining for GFRA1 (red) and TRA98 (green) in sections of PD6-8 control and E4f1 cKO testes. n=4. (F) Quantification of progenitor spermatogonia per 500 germ cells in sections of PD6-8 control and E4f1 cKO testes. n=4. (G) Immunofluorescence staining for TRA98 (green) in sections of PD3 and PD6 control and E4f1 cKO testes. n=4. (H) Percentage of germ cells located in basement membrane of PD3 (left) and PD6 (right) control and E4f1 cKO testes. n=4. Figure S2 Impact of E4f1 deletion on cell cycle progression and apoptosis. (A) Flow cytometry analysis of cell cycle in THY1+ cells from controls and E4f1 cKO testes by PI staining. (B) Histogram of cell cycle distribution of THY1+ cells from controls and E4f1 cKO testes. n=3. (C) Flow cytometry analysis of apoptotic cells in THY1+ cells from controls and E4f1 cKO testes by Annexin V staining. (D) Percentage of Annexin V positive cells in THY1+ cells from controls and E4f1 cKO testes. n=2. Error bars represent SD. *p &lt; 0.05, Student t test. Figure S3 Quality control of scRNA-seq data. (A)UMAP plot of cells from D6 control mouse testes and representative markers for each cell type. (B) UMAP plot of cells from D6 E4f1 cKO mouse testes and representative markers for each cell type. (C) Distribution of basic cell information of each sample before filtering, including the number of detected genes (Y-axis) in each sample; Distribution of the total number of UMI detected in a single cell of each sample (Y-axis);Percentage (Y-axis) distribution of mitochondrial gene expression in individual cells of each sample. Figure S4 Supplementation of H2O2 did not rescue proliferative defects of E4f1-deficient germ cells. (A) Images of MitoSOXTM red staining for THY1+ cells from controls and E4f1 cKO testes by fluorescence microscope (up) and FACS analysis (down). (B) Immunofluorescence staining of LIN28A(green) and EdU(red) in control and E4f1 cKO testicular sections supplemented with H2O2 and cultured for 2 days. -H2O2 indicates control and+H2O2 means cultured tissued were treated with H2O2.n=3. (C) Percentage of EdU+ spermatogonia in control and E4f1 cKO testicular sections supplemented with H2O2 and cultured for 2 days. n=3. Figure S5 Genotyping results of Trp53; E4f1 double knockout animals (A) Images of genotypic identification.Trp53: flox/flox 584bp; WT 431bp. E4f1: flox/flox 208bp; WT 140bp. Vasa-cre: cre 240bp; WT 324bp.