Figure 4

Erk5 expression is significantly reduced in BMDM and mature osteoclast cultures from Erk5 ^fl/fl mice compared to WT in parallel with a significant increase in osteoclast numbers and expression of the osteoclast markers, Rank, Ctsk and Nfatc1. (A) QPCR analysis of Erk5 (Exon 4) mRNA expression (normalised to housekeeping gene, Hmbs) in BMDM and osteoclast cultures generated from Erk5 ^fl/fl and WT mice. Shown are means; error bars represent SEM; t test (unpaired, 2 tailed) was used to calculate p values and those with significance are shown. *p < 0.05 from WT. (B) Western blot analysis (Left panel) of total ERK5 protein expression in BMDM cultures derived from WT (n = 5) and Erk5 ^fl/fl (n = 5) mice. Black box highlights colorimetric image of molecular weight markers which was overlaid with chemiluminescent image of samples to confirm the size of observed bands. Full images are shown in Fig. S3. Densitometry analysis (right panel) was performed using image J to normalise total ERK5 levels to those of the housekeeping gene, HSC70. Shown are mean normalised expression levels of ERK5 for WT and Erk5 ^fl/fl; error bars represent SEM; t test (unpaired, 2 tailed) was used to calculate p values and those with significance are shown. *p < 0.05 from WT. (C) Osteoclast formation is increased in RANKL-stimulated BMDM cultures derived from Erk5 ^fl/fl mice compared to WT. Figure shown is combined data from 3 independent experiments; n = 5 for each concentration of RANKL used within each experiment. Data was normalised to and is expressed as % of WT treated with 100 ng/mL RANKL. Shown are means; error bars represent SEM; ANOVA was used to calculate p values and those with significance are shown. **p < 0.01 from WT. (D) MEK5 inhibitors BIX02188 and BIX02189 (100 nM) stimulate osteoclast formation in WT cultures. Figure shown is combined data from 3 independent experiments; n = 5 for vehicle and each inhibitor used within each experiment. Data was normalised to and is expressed as % of vehicle control. Shown are means; error bars represent SEM; ANOVA was used to calculate p values and those with significance are shown. **p < 0.01 from vehicle. (E) QPCR analysis of Rank, Ctsk and Nfatc1 mRNA expression (normalised to housekeeping gene, Hmbs) in BMDM and osteoclast cultures generated from Erk5 ^fl/fl (n = 4) and WT (n = 3) mice. Shown are means; error bars represent SEM; t test (unpaired, 2 tailed) was used to calculate p values and those with significance are shown. *p < 0.05; **p < 0.05, ****p < 0.0001 from WT.