Fig 2: Impact of single and double ORMDL KOs on the activation of BMMCs. (a) Representative immunoblot and statistical analysis of total ORMDL protein levels in BMMCs derived from the indicated WT and transgenic mice. HPRT was used as a loading control. n = 6 biological replicates. Uncropped immunoblots are shown in Supplementary Fig. S5. (b) Release of β-glucuronidase as a measure of degranulation in BMMCs activated with 500 ng/ml and 1000 ng/ml TNP-BSA antigen. n = 4–11 biological replicates. Fura-2AM fluorescence as a measure of intracellular calcium at baseline and following activation with (c) 500 ng/ml TNP-BSA antigen and (d) 1000 ng/ml TNP-BSA antigen. Black arrows indicate times at which activators were added to cells. n = 4–11 biological replicates. (e) Immunoblot analysis with statistical evaluation of p-Akt, p-iκB and corresponding loading controls from lysates of non-activated BMMCs and BMMCs activated with 1000 ng/mL TNP-BSA antigen for 2 and 5 min as indicated. n = 4–14 biological replicates. Uncropped immunoblots are shown in Supplementary Figs. S6 and S7. (f) Relative mRNA expression of TNF-α, IL-6 and IL-13 in non-activated BMMCs and BMMCs activated with 1000 ng/ml TNP-BSA antigen for 1 h. n = 6–11 biological replicates. Data were analysed by one-way ANOVA with Tukey’s posttest (a, b and f), two-way ANOVA with Tukey’s posttest (c and d) and Kruskal–Wallis test with Dunn’s posttest (e). *p < 0.05, **p < 0.01 and ***p < 0.001. In (c), colour of lines and asterisks indicate statistical significance of O1/3dKO versus corresponding phenotypes—WT = grey, O1KO = yellow, O2KO = blue and O3KO = brown. In (d), black line and asterisks indicate statistical significance of O1/3dKO cells versus all other samples. All the results are represented as mean ± SEM. Ag—Antigen, Act.—Activation.

Fig 3: Impaired IκB-α phosphorylation and cellular TNF-α expression in 1-heptanol-treated BMMCs. (A) IκB-α phosphorylation (p-IκB-α) and expression of IkB-α and GRB-2 were determined by SDS-PAGE size-fractionation and IB of lysates from BMMCs treated with 2.5 mM 1-heptanol or vehicle (Control) and activated for the indicated time intervals with antigen (TNP-BSA; 0.5 µg/mL; n = 3). Representative immunoblots developed with the corresponding antibodies are shown. (B) The results from the quantification of data as in A normalized to signals in non-activated cells and loading control proteins. (C,D) RT-PCR quantification of TNF-α mRNA ((C); n = 4) and IL-6 ((D); n = 6) in non-activated or antigen-activated (Ag; 0.5 µg/mL; 1h) BMMCs pretreated for 15 min with 2.5 mM 1-heptanol or vehicle (Control). (E,F) The levels of TNF-α (E) and IL-6 (F) released into the supernatant of IgE-sensitized BMMCs pretreated with vehicle (Control; n = 4) or 2.5 mM 1-heptanol (n = 4) and non-activated or activated with antigen (Ag) for 4 h. Values indicate means ± SEM calculated from n, which show the numbers of biological replicates; ** p < 0.01, *** p < 0.001, **** p < 0.0001.