(A) Validation of RNA-seq data by northern blot using total RNA extracted from control and dHNF4 mutant adults. Affected transcripts include those involved in glucose homeostasis (Hex-C, pdgy), the electron transport chain (Sdhaf4, mt:ND1, mt:ND2, mt:ND4, mt:ND5, mt:CoxI, mt:Cox2, mt:Cox3, mt:ATPase6/8, mt:Cyt-b and mt:lrRNA), the TCA cycle (Scsalpha, dSdhaf4), and insulin signaling (4EBP, InR). rp49 is included as a control for loading and transfer. Mitochondrial-encoded transcripts are indicated by the prefix 'mt'. Depicted results were consistent across multiple experiments. (B–C) ChIP-seq analysis performed on adult flies for endogenous dHNF4 genomic binding shows direct association with both nuclear (B) and mitochondrial-encoded (C) genes involved in OXPHOS. Data tracks display q value FDR (QValFDR) significance values (y-axis) compared to input control, where QValFDR 50 corresponds to P=10–5 and 100 corresponds to P=10–10. Gene names in bold represent those expressed at reduced levels in dHNF4 mutants by RNA-seq and/or northern blot analysis. Gene names in red (ND6, Cyt-B) denote the mtDNA-encoded transcriptional unit confirmed to show no change in dHNF4 mutants. (D) Whole-mount immunostaining of adult fat body tissue for ATP5A (green) to detect mitochondria and DAPI (blue) to mark nuclei, showing fragmented mitochondrial morphology in dHNF4 mutants. (E) Analysis of dHNF4 mutant MARCM clones (GFP+) shows reduced mitochondrial membrane potential by TMRE staining of live fat body tissue from adult flies maintained on the 15% sugar diet.