Journal: Cell reports

Article Title: Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

doi: 10.1016/j.celrep.2025.116868

Figure Lengend Snippet: (A, C, and E) Representative images of whole-mount human seminiferous tubules stained with antisera against PIWIL4 (green) and the indicated markers (magenta). Nuclei were counterstained with DAPI (gray). Testicular samples from 3 fertile individuals were analyzed. Scale bars: 100 μm and 20 μm (insets). (B, D, and F) Quantification of positive cells that co-stain or not, as indicated. n , the total number of SPG counted. Boxplot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. (G) IHC analysis of PIWIL4 on human testis sections. Scale bars: 50 μm. Black arrow, A dark SPG; orange arrow, A pale SPG. (H) Quantification of A pale and A dark PIWIL4+ SPG. Testicular samples from 3 fertile individuals were analyzed. (I, K, M, and O) Representative images of whole-mount human seminiferous tubules stained with antisera against NANOS3 (yellow) and the indicated markers (magenta). Nuclei were counterstained with DAPI (gray). Testicular samples from 3 fertile individuals were analyzed. Scale bars: 100 μm and 20 μm (insets). (J, L, and P) Quantification of positive cells that co-stain or not, as indicated. n , the total number of SPG counted. Boxplot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. (N) tSNE plot showing PIWIL4 and NANOS3 expression across SPG clusters from scRNA-seq analysis. (Q) Schematic model of the human SPG compartment based on the expression of EGR4, PPP1R36, PIWIL4, and NANOS3. See also .

Article Snippet: Rabbit polyclonal anti Human NANOS3 , Proteintech , Cat# 21679-1-AP; RRID:AB_1085925.

Techniques: Staining, Expressing

Journal: Cell reports

Article Title: Decoding the heterogeneity of human undifferentiated spermatogonia reveals RAS-dependent regulation of stem cell fate

doi: 10.1016/j.celrep.2025.116868

Figure Lengend Snippet: (A) Differentially expressed genes (DEGs; q < 0.01, fold change >2) from PIWIL4+ vs. NANOS3+ cells. (B)Signaling pathways associated with the DEGs defined in (A). Statistical significance (−log10 ( p value)) is indicated by the bar. The number of DEGs for a given category is indicated by an “X.”. (C) Schematic of experimental design. Fresh human testis biopsies were processed in two ways: (1) dissociation followed by magnetic-activated cell sorting (MACS) to isolate FSD1+ uSPG for feeder-free culture with or without RAS inhibitors (RASis) or (2) maintained in transwell systems, also treated with or without RASis. (D and E) RT-qPCR analysis of feeder-free human uSPG (enriched for FSD1+ cells) cultured for 1 week and treated with or without RASis: lonafarnib (D) and salirasib (E). (F) Immunofluorescence analysis of human testicular tissue fragments cultured with or without RASis, stained for PIWIL4, NANOS3, and BrdU. Cell nuclei were counterstained with DAPI (gray). Scale bars: 50 μm. (G and H) Quantification of PIWIL4+ ( n = 328) and NANOS3+ ( n = 431) per cross-section across four samples. (I and J) BrdU index of PIWIL4+ (I) and NANOS3+ (J) cells in cultured testicular fragments, treated with or without RASis. Data are represented as the mean ± SEM. Four biological replications were performed. For (D), (E), and (G)–(J), data are represented as the mean ± SEM. Four biological replications were performed. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not statistically significant, as determined by unpaired t test (D and E) or one-way ANOVA (G–J).

Article Snippet: Rabbit polyclonal anti Human NANOS3 , Proteintech , Cat# 21679-1-AP; RRID:AB_1085925.

Techniques: Protein-Protein interactions, FACS, Quantitative RT-PCR, Cell Culture, Immunofluorescence, Staining