2.1 Cell culture of TM3 cell line

The mouse Leydig cell line TM3 was obtained from Shanghai Yuchi Biotech Co., Ltd. (SC0443) and authenticated via short tandem repeat (STR) profiling to confirm species origin. Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, C11995500BT, Suzhou, China) supplemented with 10% fetal bovine serum (FBS, Lonsera, S711-001S, Montevideo, Uruguay) and 1% penicillin-streptomycin solution (Servicebio, G4003, Wuhan, China). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂, and the culture medium was refreshed every 2–3 days. Upon reaching approximately 80% confluence, cells were detached using trypsin (Beyotime) and subcultured at a 1:3 ratio. For experimental treatments, TM3 cells were exposed to various concentrations of Pyrotinib, with control cells receiving 0.1‰ dimethyl sulfoxide (DMSO) as the vehicle control. Detailed protocols for drug administration, sample collection, and downstream analyses are described in the corresponding sections.

2.2 Preparation of Pyrotinib solution

Pyrotinib, provided as an active pharmaceutical ingredient by Jiangsu Hengrui Medicine Co., Ltd. (H20180012, Lianyungang, Jiangsu, China), was initially dissolved in dimethyl sulfoxide (DMSO) to prepare a 0.1 M stock solution. The stock solution was aliquoted and stored appropriately to prevent degradation. Prior to use, the stock was diluted with TM3 culture medium to the required working concentrations for experimental treatments, ensuring the final DMSO concentration in all treatment groups did not exceed 0.1‰.

2.3 Clonogenic assay

TM3 cells were cultured until they reached 80–90% confluence and then subjected to different treatments. Cells in the experimental group were treated with 100 nM pyrotinib, while those in the control group received an equivalent volume of 0.1‰ DMSO. After 24 hours of treatment, cells were trypsinized, counted and seeded into 6-well plates at a density of 500 cells per well. The cells were then cultured under standard conditions for 14 days, with medium changed every 2–3 days. At the end of the incubation period, colonies were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.5% crystal violet for 20 minutes. Colony formation was subsequently visualized and assessed under a microscope.

2.4 Cell viability assay

TM3 cells in the logarithmic growth phase were trypsinized, counted and seeded into 96-well plates at a density of 3000 cells per well. Following 24 hours of incubation to allow for cell attachment, the cells were treated with Pyrotinib at six different concentrations: 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM and 10,000 nM. Each concentration group included three replicate wells. After an additional 24-hour exposure period, cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Yisheng Biotech, 40203ES76, Shanghai, China), following the manufacturer’s protocol. The CCK-8 reagent was added to each well and incubated for 1 hour at 37 °C. Absorbance was measured at 450 nm using a microplate reader. The data were used to construct a dose—response curve for Pyrotinib-induced cytotoxicity.

2.5 Cell cycle analysis

TM3 cells in the logarithmic growth phase were harvested, counted, and seeded into 6-well plates at a density of 5 × 10⁵ cells per well. After 24 hours of incubation, cells in the experimental group were treated with 100 nM Pyrotinib, while control cells received 0.1‰ DMSO. Following a 48-hour treatment period, cells were collected and fixed in 75% ethanol at −20 °C for at least 24 hours. Cell cycle staining was performed according to the manufacturer’s instructions using a commercial cell cycle analysis kit (Lianke Biotechnology, CCS012, Hangzhou, China). Data acquisition was carried out on a CytoFLEX flow cytometer (Cytoflex LX, Beckman Coulter, Brea, CA, ​USA), and cell cycle distribution was analyzed accordingly.

2.6 Western-blot assay

TM3 cells in the logarithmic growth phase were seeded into 6-well plates at a density of 2 × 10⁵ cells per well. After 24 hours, cells were treated with Pyrotinib at concentrations of 0 nM, 50 nM and 100 nM. Total protein was extracted at 2, 24 and 48 hours post-treatment using RIPA lysis buffer (Beyotime, P0013B, Shanghai, China), and protein concentrations were determined using a bicinchoninic acid (BCA) assay kit (Beyotime, P0010S, Shanghai, China). Equal amounts of protein were separated on 10% sodium dodecyl sulphate (SDS)—polyacrylamide gels (1.5 mm, 15-well; Epizyme) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Membranes were blocked with QuickBlock™ blocking buffer (Beyotime, P0252, Shanghai, China) and incubated with specific primary antibodies (see Table 1 for details). Detection was performed using appropriate secondary antibodies and visualized with the Bio-Rad Gel Imaging System.

2.7 Transcriptome sequencing

TM3 cells in the logarithmic growth phase were dissociated and counted, then seeded into 6-well plates at a density of 500,000 cells per well. The treatment group received 100 nM Pyrotinib, while the control group was treated with 0.1‰ DMSO, with three replicates for each group. After 48 hours of treatment, RNA was extracted using TRIzol reagent (Takara, 9108, Shiga, Japan) and sent to Beijing Novogene Co., Ltd. for transcriptome sequencing. RNA sequencing was performed on the Illumina Novaseq platform, generating approximately 150 bp paired-end reads per sample. RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA) with the RNA Nano 6000 Assay Kit (5067-1511, Agilent, CA, USA). Total RNA was utilized to construct libraries through poly-A selection, followed by fragmentation, cDNA synthesis with random hexamer primers, and adaptor ligation according to the manufacturer’s protocols (Agilent, Santa Clara, CA). The libraries were quantified via quantitative polymerase chain reaction (qPCR) with a KAPA Library Quantification Kit (Kapa Biosystems, KK4824, Woburn, MA, USA), and clustering was conducted on an Illumina cBot system using the TruSeq PE Cluster Kit v3-cBot-HS (PE-401-3001, San Diego, CA, USA).

2.8 Bioinformatic analyses

Data preprocessing was performed on the raw fastq files using fastp (v0.20.0, HaploX Biotechnology, Shenzhen, China) [9] to remove low-quality reads, reads with poly-N and adapter sequences, resulting in high-quality reads (Q20, Q30). Clean reads were aligned to the reference genome using Hisat2 (v2.0.5, Center for Computational Biology, Johns Hopkins University, Baltimore, MD, USA) [10], with gene annotations in Gene transfer format (GTF) format. Hisat2 was chosen for its ability to handle splice junctions, allowing for high-quality alignments. Reads with mapping quality of less than 30 were filtered out.

For gene expression quantification, the featureCounts tool (v1.5.0-p3, Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia) [11] was used to count the reads mapped to each gene, and the FPKM (Fragments Per Kilobase of transcript per Million mapped reads) method was applied to normalize gene expression levels across samples.

Principal component analysis was performed using the FactoMineR package (v2.11, Agrocampus Ouest, Rennes, France) [12] and visualized with the factoextra package (v1.0.7, Marseille, France) to explore the overall structure of the data and visualize sample clustering. Differential expression analysis was conducted between the two groups, Pyrotinib (Py) and normal control (NC), each with three biological replicates. The DESeq2 package (v1.20.0, European Molecular Biology Laboratory, Heidelberg, BW, Germany) [13] in R was used to identify significantly differentially expressed genes, with significance defined by an adjusted p-value (padj) ≤ 0.05 and an absolute Log2 fold change (∣Log2 (FoldChange)∣ ≥1). Gene Ontology (GO) [14] and Kyoto Encyclopedia of Genes and Genomes (KEGG) [15] enrichment analyses were carried out using the clusterProfiler (v3.10.0, Southern Medical University, Guangzhou, China) [16], applying a corrected padj ≤ 0.05 to determine significantly enriched terms and pathways.

All downstream analyses, including differential expression, visualization and enrichment analyses, were conducted in R (v3.5.0, R Core Team, R Foundation for Statistical Computing, Vienna, Austria).

3.1 Pyrotinib inhibited the proliferation of TM3 cells

To evaluate the effects of Pyrotinib on the male reproductive system, TM3 mouse Leydig cells were employed as an in vitro model. Following 24 hours of treatment, a significant increase in apoptosis was observed in the Pyrotinib-treated group compared to the control group, as determined by flow cytometry (Fig. 1A). Clonogenic assays further demonstrated a marked decrease in colony-forming ability upon Pyrotinib exposure (Fig. 1B). A dose-dependent reduction in cell viability was also noted, with an estimated half-maximal inhibitory concentration (IC₅₀) of approximately 95.74 nM (Fig. 1C). Cell cycle analysis revealed that Pyrotinib treatment led to cell cycle arrest at the G1 phase, with 67.2% of cells in the treated group remaining in G1 compared to 58.3% in the control group (Fig. 1D,E). Collectively, these findings indicate that Pyrotinib significantly impairs the proliferative capacity and viability of TM3 cells, primarily by inducing apoptosis and arresting cell cycle progression at the G1 phase, thereby inhibiting entry into the S phase.

Fig. 1.Effects of Pyrotinib on TM3 cell proliferation and cell cycle progression. (A) Pyrotinib treatment (100 nM, 24 h) significantly increased apoptosis in TM3 cells compared to the control group, as shown by representative images. Scale bar: 100 µm. (B) Colony formation assay showing a marked reduction in the number of colonies after 24 hours of Pyrotinib treatment, visualized by crystal violet staining. (C) Cell viability decreased in a dose-dependent manner with increasing concentrations of Pyrotinib; IC₅₀ was approximately 95.74 nM (n = 6). (D) Flow cytometry analysis revealed that 67.2% of TM3 cells in the Pyrotinib-treated group were arrested in the G1 phase, compared to 58.3% in the control group (n = 3). (E) Quantitative comparison of cell cycle distribution between the Pyrotinib-treated and control groups (n = 3). Data are presented as mean ± standard deviation. Abbreviations: RMSD: root mean square deviation; IC₅₀: half maximal inhibitory concentration; G0: Gap 0; G1: Gap 1; G2: Gap 2; S: Synthesis; CV: coefficient of variation.

3.2 Pyrotinib modulates PI3K/AKT and MAPK/ERK signaling pathways in TM3 cells

To investigate the molecular mechanisms underlying the Pyrotinib-induced reduction in proliferation and viability of TM3 cells, Western blot analysis was performed. This examined the expression levels of key signaling proteins, including AKT, ERK, phosphorylated AKT (p-AKT), phosphorylated ERK (p-ERK) and phosphorylated S6 kinase (p-S6K). As shown in Fig. 2A,B (and Supplementary Fig. 1), short-term treatment with Pyrotinib (2 hours) led to a transient increase in the phosphorylation levels of these proteins. However, prolonged exposure to Pyrotinib (24 to 48 hours) resulted in a marked decrease in the phosphorylation levels of proteins involved in both the PI3K/AKT and MAPK/ERK signaling pathways. These results suggest that Pyrotinib may initially activate, but subsequently suppress, key proliferative and survival pathways in TM3 cells, thereby contributing to its inhibitory effects on cell growth and viability.

Fig. 2.Effects of Pyrotinib on PI3K/AKT and MAPK/ERK Signaling Pathways in TM3 Cells. (A) Western blot analysis showing the expression levels of total and phosphorylated AKT, ERK and S6K following treatment with different concentrations of Pyrotinib. A transient increase in phosphorylated protein levels was observed at early time points. (B) Quantitative analysis of phosphorylated protein expression (p-AKT, p-ERK and p-S6K) at various time points, illustrating a significant reduction after 24 and 48 hours of Pyrotinib exposure. Data are presented as mean ± standard deviation (n = 3). Abbreviations: AKT: Ak strain transforming; ERK: extracellular signal-regulated kinase; p-AKT: phosphorylated AKT; p-ERK: phosphorylated ERK; p-S6K: phosphorylated S6 kinase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: normal control; Py: Pyrotinib-treated group.

3.3 Transcriptomic analysis reveals molecular mechanisms underlying Pyrotinib treatment

To elucidate the molecular mechanisms mediating the effects of Pyrotinib on TM3 cells, transcriptome sequencing was conducted on samples collected prior to treatment and at 48 hours post-treatment. Principal component analysis (PCA) demonstrated clear sample clustering, with confidence ellipses encompassing all replicates within one standard deviation of the mean (Fig. 3A). Differential gene expression analysis, visualized via a volcano plot, identified 170 significantly upregulated and 197 downregulated genes following Pyrotinib exposure (Fig. 3B). Gene Ontology (GO) enrichment analysis revealed that the downregulated genes were predominantly associated with biological processes related to chromatin dynamics and mitosis, including mitotic spindle assembly, mitotic sister chromatid segregation and regulation of chromosome segregation (Fig. 3C). KEGG pathway analysis further indicated significant enrichment of these genes in the cell cycle pathway (Fig. 3D). Collectively, these data suggest that Pyrotinib exerts its antiproliferative effects in TM3 cells, at least in part, through the downregulation of key genes involved in cell cycle progression and mitotic processes.

Fig. 3.Transcriptomic and enrichment analyses following Pyrotinib treatment in TM3 cells. (A) Principal component analysis (PCA) plot illustrating the distribution of samples based on the first two principal components. Each dot represents a sample, colored by group: NC (normal control, teal) and Py (Pyrotinib-treated, yellow). (B) Volcano plot displaying differentially expressed genes between Py and NC groups. Significantly downregulated genes are shown in blue, and upregulated genes in red (adjusted p < 0.05, (∣Log2 (FoldChange)∣ ≥ 1). (C) Gene Ontology (GO) enrichment analysis of downregulated genes, showing significantly enriched biological process terms. Dot size indicates the number of genes annotated to each term, while color intensity reflects the statistical significance (adjusted p value). (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of downregulated genes. Enriched pathways are presented similarly to GO terms with respect to dot size and significance. Abbreviations: PC1: the first principal components; PC2: the second principal components.