Fig 2: DDX5 regulates cell cycle progression in spermatogonia post-transcriptionally. a Pathway analysis of differentially expressed genes identified by RNA-sequencing comparing control and Ddx5-ablated cultured spermatogonia. b Differentially expressed cell cycle-related genes identified by RNA-sequencing comparing control and Ddx5-ablated cultured spermatogonia. Fold change in Ddx5-ablated spermatogonia shown relative to control. Mean ± SEM shown; n = 4 independent biological replicates per condition. # denotes FDR <0.05. c Representative flow cytometry plots (left) showing Ddx5 loss results in cell cycle arrest in cultured undifferentiated spermatogonia at D1 post-ablation. Control: vehicle-treated control spermatogonia; Ddx5TAM-KO: tamoxifen-induced Ddx5-ablated spermatogonia. Representative of n = 3 independent experiments. Histogram (right) depicts quantification of cell proportions for each cell cycle phase determined by flow cytometry. *P < 0.05; **P < 0.01. Two-tailed unpaired t-test, n = 3 independent experiments per condition. d Representative western blot showing reduced CCNA2, CCND1 and CCND2 protein upon loss of Ddx5 (Ddx5TAM-KO) compared with control in cultured undifferentiated spermatogonia at D2 post-tamoxifen treatment. Representative of n = 3 independent experiments. e Quantification of western blots represented in d. Mean ± SEM shown; *P < 0.05; **P < 0.01; Two-tailed unpaired t-test. n = 4 independent samples per condition. f RNA immunoprecipitation using DDX5 antibody in cultured wildtype undifferentiated spermatogonia followed by RT-qPCR for cyclin genes. Fold enrichment versus IgG control shown. Mean ± SEM shown; n = 2 independent experiments. g Nuclear-cytoplasmic ratio of Ccnd1 and Ccnd2 transcripts determined by RT-qPCR of control and Ddx5-ablated (Ddx5TAM-KO) cultured undifferentiated spermatogonia subcellular fractions. Fold change relative to control shown. Mean ± SEM shown; *P < 0.05; Mann–Whitney U test; n = 4 independent samples per condition. h Analysis of mRNA stability over time following inhibition of transcription by actinomycin D treatment in control versus Ddx5-ablated (Ddx5TAM-KO) cultured undifferentiated spermatogonia. mRNA expression determined by RT-qPCR (normalised to Actb) relative to initial levels at time 0 h. *P < 0.05; Two-way ANOVA with Bonferroni multiple comparisons test; mean ± SEM shown; n = 4 independent samples per condition