Fig 2: Characterization of antibodies used for quantification of protein acetylation in honeybee brain.(A) The antibodies against histone H3, H3K9ac, H3K18ac and acetylated lysine were tested on Western blots with separated protein from honeybee brain. All antibodies against H3 (and modifications) stain a single band at the molecular weight of H3. (B, C, D) Immunolabeling of the antigens recognized by antibodies against H3K9ac (B), H3K18ac (C) and H3 (D). The antibodies stain all somata in the honeybee brain. (C1) The higher magnification shows that labeling is restricted to the nuclei. Depicted are Kenyon cells of the mushroom bodies (MB). The brain area used for quantification of the acetylation status (dashed lines in B) contains somata from the central brain, antennal lobes, suboesophageal ganglia (SOB) (all together˜20%), and soma from Kenyon cells of the mushroom bodies (˜80%). The optical lobes (OL), the retina (Re) and the ocelli (Oc) were excluded from the measurements.

Fig 3: Exposure of cumulus cells to ß-hydroxybutyrate (BOHB) does not alter acetylation levels but upregulate the “fasting” response gene PPARA. (A) Diagram illustrating the exposure of cumulus-oocyte complexes (COCs) to BOHB. After BOHB treatment during in vitro maturation, the oocytes were stripped off and the cumulus cells were used for western blots and gene expression analysis. (B) Immunoblots for H3K9ac, total histone H3 (H3) and histones stained with Coomassie Blue. (C) Quantification of H3K9ac levels in cumulus cells from COCs maturated alone (negative control) or in the presence of BOHB (2 mM) and TSA (100 nM; positive control) for 21–23 h. H3K9ac levels were calculated in relation to total H3. (D) Relative mRNA abundance of PPARA in control and BOHB-treated cumulus cells. Data are presented as fold change in relation to the control group, and values are shown as the mean ± s.e.m. Gene expression data from 6 biological replicates. Different letters indicate a significant difference with respect to the control group (P < 0.05). The western blot images were cropped for illustrative purposes. The full gels/blots are presented in Supplemental Fig. 7.