Fig 2: Co-localization of EHBP1L1 and ACTIN during the first wave of rat spermatogenesis (7–28 dpp). (A,D,G,J) DAPI-fluorescent nuclear staining (blue) and ACTIN staining (red). (B,E,H,K) EHBP1L1 fluorescence (green). (C,F,I,L) Merged fluorescent channels (blue/red/green). The intermediate yellow–orange tint indicates EHBP1L1 and ACTIN co-localization. (A–C) 7 dpp testis; (D–F) 14 dpp; (G–I) 21 dpp; (J–L) 28 dpp. (M–P) Negative controls for the same time points, obtained by omitting the primary antibodies. Scale bars represent 20 μm, except for the insets, where they represent 10 μm. Arrowhead: gonocytes; striped arrowhead: SPG; arrow: I SPC; dotted arrow: rSPT.

Fig 3: Therapeutic potential of EHBP1L1 knockdown and anti‐PD‐1 combination therapy in RCC PDX models. A) Schematic diagram of the generation and adoptive transfer of human RCC‐specific CD8+ T cells into NCG mice transplanted with RCC PDXs. B) Schematic diagram showing that RCC PDX mice were treated with adoptive T cell transfer, anti‐PD‐1 antibody and siRNA at the indicated time points. In the figure, ‐21 indicates the day of subcutaneous inoculation of RCC PDXs. C–F) The anti‐PD‐1 and siEHBP1L1 combination therapy synergistically suppressed the growth of tumors in RCC PDX mice (n = 5 per group). C) Tumor growth curves, D) tumor images, E) tumor weights, and F) survival curves. G) Flow cytometry analysis of IFN‐γ, TNF‐α, GZMB, and Perforin in CD8+ T cells isolated from PDX tumors with the indicated treatments. H) ELISA analysis of the IFN‐γ and TNF‐α levels in serum samples from PDX mice with the indicated treatments. I) Representative flow cytometry histograms (left panel) and statistical analysis (right panel) of cell surface PD‐L1 in tumors from PDX mice with the indicated treatments. J) Representative IHC staining images of JAK1 in tumors from PDX mice with the indicated treatments (left panel) and statistical analysis (right panel) of IHC scores. Scale bar = 100 µm. C–F) Data represent one independent experiment with 5 mice per group. G–J) Each experiment was repeated three times with 5 mice per group, and data shown are representative of three independent experiments.

Fig 4: Localization of EHBP1L1 in adult rat testis (60 dpp). (A) EHBP1L1 localization in different stages of adult rat spermatogenesis. To better show the differences between them, the 14 stages comprising the whole spermatogenic cycle are arbitrarily sub-divided into four parts. Scale bars represent 20 μm. Arrow: I SPC; dotted arrow: rSPT; striped arrow: eSTP. (B) EHBP1L1 localization in SPT in comparison with a schematic representation of the different phases of acrosome biogenesis. In A and B merged images, the blue channel represents DAPI-fluorescent nuclear staining, the red channel represents PNA lectin acrosome staining, and the green channel represents EHBP1L1 fluorescent signal.

Fig 5: EHBP1L1 protein level during the first wave of rat spermatogenesis. (A): Western blot analysis showing the expression of EHBP1L1 (164 kDa, top section) and ACTIN (42 kDa, bottom section) during rat testis post-natal development at 7, 14, 21, 28, 35, 42, and 60 days postpartum (dpp). (B): Histogram showing the relative expression levels of EHBP1L1 in all the analyzed samples. Data were normalized with ACTIN and reported as EHBP1L1/ACT OD ratio. All data represent the mean ± SEM. a vs. b: p < 0.01. Western blot experiment was performed in triplicate.