Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P < 0.005 (one-way ANOVA with Tukey’s HSD post hoc test). (B) Merged DIC and fluorescence images of a single spermatogonium in culture that resides on Sertoli cells and is targeted by a patch pipette (pp). FITC-labeled BLOCK-iT fluorescent oligos allow identification of cotransfected cells by their fluorescence. (C) Original current density traces depicting representative whole-cell voltage-clamp recordings from spermatogonia exposed to ATP (300 µM; 1 s; V_hold = −80 mV; S₁, S₆). siRNA transfection, or the lack thereof (black), is color coded for nontargeting constructs (gray), two different targeting siRNAs (green), and a control construct targeting P2X2 expression (red). (D) Bar diagram quantifying the results exemplified in C. Data are means ± SEM. Numbers of experiments are denoted above bars. Color code as in C. Asterisks (*) indicate statistical significance, P < 0.05 (one-way ANOVA with Tukey’s HSD post hoc test).