Fig 2: Alignment of the consensus sequences of p38, JNK, Hog1 and the homologs from unicellular opisthokonts. Alignment colour-codes correspond to AA identity, from the highest (dark blue) to low (white, less than 40% of positions in a column is conserved). Designation: (+) ATP binding site; (s) substrate binding site; (o) AA interacting with the conserved threonine when phosphorylated, necessary for folding into active state conformation; (d) docking site, (L) lipid binding site; (!) hinge point, the two domains rotating around hinge point during activation; asterisks mark the phosphorylation sites specific to the stress MAPKs. Blue circles mark sites shared by two or more paralogs, red circles mark regions that can distinguish between p38, JNK and Hog1. Parvul—sequence from P. atlantis, Fil-Icht—consensus sequence for the clade filastereans + ichthyosporeans from the tree in figure 1, Creolim—sequence from Creolimax fragrantissima, Corallo—sequence from Corallochytrium limacisporum, Choan1 and Choan2—consensus sequences from choanoflagellates choano-1 and choano-2, respectively. Kinases: p38a (MAPK14), p38ß (MAPK11), p38? (MAPK12) and p38d (MAPK13).

Fig 3: LrB inhibits MAPK and NFATc1 signaling pathways. (A) BMMs were seeded in 6-well plates and cultured with culture medium until they reached 90% confluence. LrB was used to pretreat the cells for 1 hour followed by 0, 5, 10, 20, 30 and 60 minutes of RANKL stimulation. Total cellular proteins were extracted using RIPA lysis buffer and cell lysates were analyzed by Western blotting using primary antibodies specific to p-p38 and p38, p-JNK and JNK. Representative images showed the inhibition effect of LrB on MAPK pathway signaling specific to p-p38 and p-JNK. (B) Quantitative analyses of p-p38 and p-JNK were normalized to total p38 and JNK respectively. Phosphorylation levels of p38 and JNK were significantly suppressed by LrB from 5 minutes to 30 minutes. (C) NFATc1 transcriptional activity was measured by a luciferase reporter gene assay. RAW264.7 cell line was stably transfected with an NFATc1 luciferase reporter gene. RAW-NFAT-luc cells were seeded in 48-well plate and pretreated with LrB with different concentrations for 2 hours, then stimulated by RANKL for 24 hours. Cells were then lysed by lysis buffer and luciferase activity was measured using a luciferase reporter assay kit. Quantitative analyses indicated that LrB inhibits NFATc1 transcriptional activity dose-dependently. (D) Freshly isolated BMMs were seeded in 6-well plates at the concentration of 1x105 cells per well. The cells were stimulated with RANKL on day 0, 1, 3 and 5 in the presence or absence of LrB at 10 µM. Primary antibodies including NFATc1, c-Fos, V-ATPase-d2 and CTSK were applied and ß-actin was used as a normalized control protein. Representative images were used to indicate the LrB's inhibitory effect on NFATc1 pathway activities. (E) Quantification of the ratios of band intensity of NFATc1, c-Fos, V-ATPase-d2 and CTSK relative to ß-actin. All bar charts are presented as mean ± SD; n=9. *p<0.05, relative to non-treatment group.