Fig. 4: Fat body cell death in Nacα mutants is progressive during larval development.

a, b Fat body cell death in Nacα mutants during larval development. Fat bodies of the indicated genotypes were dissected from each stage and stained for Cas3, F-actin, and DNA (a). Quantification of the nuclear diameter in fat body cells (b). E1st, early first instar; E2nd, early second instar; E3rd, early third instar; M3rd, mid-third instar (24 h after E3rd). c, d Fat body cell death in Nacα knockdown larvae. Fat bodies were dissected daily and stained for cDcp1, F-actin, and DNA (c). As the Nacα knockdown larvae delayed the development, average larval stages of the knockdown animals are indicated. DAE, days after eclosion; ML3rd, mid-late third instar before the wandering stage; L3rd, late third instar. Quantification of nuclear diameter in fat body cells (d). Stages in the parentheses indicate larval stages in control animals. ND, not determined due to puparium formation. e, f Transient knockdown of Nacα at the third instar is sufficient to induce fat body cell death. The experimental scheme for the temperature shift is shown on the left. Early third instar larvae grown at 18 °C were collected and maintained at either 18 °C or 29 °C for 24 h. Fat bodies were dissected and stained for cDcp1, F-actin, and DNA (e). Quantification of the nuclear diameter in cDcp1-negative (–) or -positive (+) fat body cells (f). g Relative gene expression levels of Nacα/β and Diap1. Fat bodies (FB) of the control larvae were dissected from the indicated stages and subjected to qRT-PCR analysis. h Genetic interaction of Diap1 with Nacα. Fat bodies of the indicated genotypes were dissected from each stage and stained. Nacα ^-/-, Nacα¹/Nacα⁰⁴³²⁹ mutants. Quantification of the nuclear diameter and cDcp1 signals is shown in Supplementary Fig. 3f, g. Horizontal lines indicate the means of individual groups (b, d, f). Values of n indicate the number of nuclei (b, d, f) from multiple animals. For appropriate panels, results are presented as the mean ± SD, n = 4 batches (g); unpaired two-tailed Welch’s t-test (b, d, f); unpaired two-tailed Student’s t-test (g). Scale bars, 50 μm (a, c, e, h).