Fig. 1.

Knockdown of CENP-A and ATP synthase F₁ subunits in testis. a Cartoon of the typical nuclear morphology of meiotic prophase I S1/2a and S5/6 stage spermatocytes showing autosomal and sex chromosome territories (grey) and associated centromeres (black foci). Timing of the bam-GAL4 driven RNAi is indicated. b Immuno-fluorescent micrograph of control (isogenic) S5/6 nuclei or nuclei RNAi-depleted of CENP-A (at 25 °C) stained with antibodies against CENP-A (red) and CENP-C (green) (n = 3). DNA is stained with DAPI (blue). Numbers indicate centromere foci per nucleus; inset shows two spots (indicated by white arrow) typically counted as two individual centromere foci. Scale bar = 10 μm. c Quantitation of centromere foci in control nuclei or nuclei RNAi-depleted of CENP-A (at 25 °C) at S1/2a, S5/6, prometaphase (PMI) or interphase stages of meiosis I. Data pooled from two independent experiments, 50 nuclei quantified per experiment. Error bars = SEM. The data were analysed using an unpaired Student's t-test, ****p < 0.0001, NS = not significant, p > 0.05. d Silver-stained SDS-PAGE gel showing input, GST only and GST-Nterm-CENP-A pull-down fractions. Arrows indicate molecular weights of GST and GST-Nterm-CENP-A in kilodaltons (kDa). e Quantitation of cysts at respective cell cycle stages (meiosis I, II or spermatids) in wild type (TRiP isogenic) or bam-Gal4 control adult testes (n = 18) or testes in which ATPsyn-α (n = 17), -β (n = 15), -βlike (n = 13) and –γ (n = 12) is RNAi-depleted at 25 °C or 29 °C. Data pooled from two individual RNAi experiments; significance tests were carried out using pooled controls (wild type and bam-Gal4). Error bars = SEM. The data were analysed using an unpaired Student's t-test, NS = not significant. f Bright-field micrograph of 5 day old control adult testis (bam-Gal4) or testis RNAi-depleted of ATPsyn-α, -β, βlike and –γ at 25 °C (n = 3). Arrowheads indicate seminal vesicles and arrows indicate abnormal testis morphology. Scale bar = 500 μm