Fig. 6.

Ilf3 loss reduces proliferation and disrupts cyclin expression in spermatogonia. a Immunofluorescence showing ILF3 is expressed in PLZF-positive cultured undifferentiated spermatogonia. Scale bar = 100 μm. b Immunofluorescence showing ILF3 is co-localised to DDX5-expressing cells within the murine seminiferous tubule. Scale bar = 50 μm. c RNA immunoprecipitation using ILF3 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. d Doxycycline (DOX)-inducible CRISPR/Cas9-mediated Ilf3 ablation in cultured undifferentiated spermatogonia shows efficient loss of ILF3 at D4 post-DOX treatment (+DOX) compared with control cells without DOX treatment (-DOX). sgRNA constructs targeting Ilf3 are GFP-tagged, allowing visualisation of spermatogonia with an anti-GFP antibody. Scale bars = 100 μm. e Loss of Ilf3 in cultured undifferentiated spermatogonia results in reduced cell numbers at D2 post-DOX treatment compared with DOX-treated wildtype control cells. ***P < 0.001. Two-tailed unpaired t-test, n = 6 replicates per condition. Mean ± SD shown. f RT-qPCR confirms downregulation of Ilf3 upon CRISPR/Cas9-mediated ablation (Ilf3^−/−) compared with control in cultured undifferentiated spermatogonia at D4 post-DOX treatment. Tested cyclin genes showed no significant difference. Cell samples were FACS-purified according to GFP to isolate undifferentiated spermatogonia prior to RNA extraction. *P < 0.05; Mann–Whitney U test, n = 4 independent samples per condition Mean ± SEM shown. g Western blot depicting reduced CCND1 protein upon CRISPR/Cas9-mediated Ilf3 ablation in cultured undifferentiated spermatogonia at D4 post-DOX treatment. Representative of n = 3 independent samples per condition (left). Quantification of CCND1 protein in control versus Ilf3-ablated (Ilf3^−/−) spermatogonia (right). Mean ± SEM *P < 0.05; Two-tailed unpaired t-test; n = 3 independent samples per condition