Fig. 6

The FICD augments NFATc1 expression by activating the MNK1/2/eIF4E axis. a, b BMDMs from WT and FICDtg^M mice were stimulated with RANKL (50 ng·mL⁻1) for the indicated times. a RT-qPCR analysis of Nfatc1 mRNA normalized to Hprt mRNA. b Immunoblot analysis with anti NFATc1, HA, or α-tubulin antibodies. c, d BMDMs from c-FMS^f/+ Mx1-Cre mice were transduced with lentiviral particles encoding FMS^wt or FMS^mut and then cultured with M-CSF and RANKL. c Immunoblot analysis of whole cell lysate with anti-NFATc1 antibody. α-Tubulin was used as a control. The left panel shows representative images. The right panel shows the cumulative intensity of NFATc1 bands. The intensity of NFATc1 in FMS^mut was set as 100%. d The mRNA expression level of NFATc1. e Immunoblot analysis of whole cell lysates with phospho-eIF4E antibodies. HA-tagged FICD was detected by HA-antibodies. α-Tubulin was used as a control. The left panel shows representative images. The right panel shows the cumulative percentage of the intensity of the band (at 24 h) relative to the control from three independent experiments. f, g BMDMs from WT mice were treated with CPG57380 at the indicated doses and then cultured with RANKL for 1 day. D: DMSO. f Immunoblot analysis with anti-NFATc1, phospho-eIF4E, or α-tubulin antibodies. The left panel shows representative images. The right panel shows the cumulative percentage of the intensity of the band relative to the control (RANKL + DMSO) (n = 4). g Nfatc1 mRNA expression was measured by qPCR relative to Hprt mRNA. the DMSO-treated RANKL condition was set as 100%. h Osteoclastogenesis assay. BMDMs from WT and FICDtg^M mice were treated with CPG57380 at the indicated doses and then cultured with RANKL for an additional 3 days. The upper panel shows representative images of TRAP-stained cells. The bottom panel shows the percentages of TRAP-positive multinuclear cells (MNCs: more than three nuclei) per control from three independent experiments. Scale bar: 100 μm. i Cell viability assay. BMDMs from WT and FICDtg^M mice were stimulated with CPG57380 at the indicated doses for 1 day. j–n K/BxN serum transfer-induced arthritis model. Nine-week-old male C57BL/6 J mice received K/BxN serum on days 0 and 2. Vehicle or CPG57380 (40 mg·kg⁻¹, CPG) was administered intraperitoneally (i.p.) from day 2 until day 13. j Schematic diagram showing the experimental design. k Ankle thickness. l Arthritis score. m Representative images of TRAP-stained histological sections from the calcaneocuboid and tarsometatarsal joints. Scale bar: 1 mm. n Histomorphometric analysis of tarsal bones. N.OC/B.Pm Osteoclast number/bone parameter. OC.S/BS osteoclast surface/bone surface. ES/BS Eroded surface/bone surface. All data are shown as the mean ± SEM. CTL, Control. *P < 0.05; n.s. not significant by one-way ANOVA with a post hoc Tukey test (a, f–i, k, l) or two-tailed, unpaired t-test (c, d, e, n). The data represent at least three experiments.