Fig 1.
Desmin staining of primary human myoblasts (20x magnification).
Most of the cells stained positive for desmin, suggesting that these are myogenic cells. Bar = 100 µm.
Fig 2.
Cell viability (MTT) assay for four conditions - media control and three concentrations of IL-1
β (1 ng/ml, 10 ng/ml, 100 ng/ml) after 72 hrs of treatment (n = 3 biological replicates, n = 8 technical replicates, media vs treatment groups is not significant, p = ns). Data shown as mean ± SEM.
Fig 3.
β triggers IL-6 production in SkMC myoblasts. Treatment of SkMCs myoblasts with IL-1β increases IL-6 compared to media alone. We observed a significant increase of IL-6 after 48h and with both 10 and 100 ng/ml of IL-1β (p < 0.05) (n = 2 biological replicates, n = 8 technical replicates). Individual graphs for each timepoint and dose are provided in the supplementary data (Fig S2 and Tables S15-22; and Fig S3 and Tables S23-30, respectively).
Fig 4.
Cell viability analysis after canakinumab treatment for 72 hr.
MTT assay for ten conditions- media control, six concentrations of canakinumab (0.001 nm, 0.01 nm, 0.1 nm, 1 nm, 10 nm, 100 nm) and three concentrations of 1 hr heat-shocked canakinumab (1 nm, 10 nm, and 100 nm) after 72 hrs of treatment (n = 2 biological replicates, n = 8 technical replicates, p = ns).
Fig 5.
IL-6 ELISA measurements for 12 treatment conditions (media controls, canakinumab (0.001 nm, 0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 250 nM, 500 nM, and 1000 nM), and 1-hr 100 nM heat-shocked canakinumab stimulated with IL-1
β using two treatment schedules for 48 h. The left graphs show 30 min pre-treatment with canakinumab before administering 10 ng/ml of IL-1β for 48 hrs. The right graphs indicate treatment with a 30 min co-incubation of canakinumab with 10 ng/ml of IL-1β before administration of solution to myoblasts for 48 hrs. A) Measured IL-6 (ng/ml) per treatment condition. B) IC50 calculation using 8 different concentrations of canakinumab treatment. For all assays shown above, n = 2 biological replicates, n = 4 technical replicates.
Fig 6.
Cell viability (MTT) assay for four conditions - media control, IL-1
β 10 ng/ml, Canakinumab 10 nM, and the co-incubation of IL-1β 10 ng/ml and canakinumab 10 nM after 72 hrs of treatment (n = 2 biological replicates, n = 3 technical replicates). Data shown as mean ± SEM.
Fig 7.
Evaluation of the effects of canakinumab in reducing IL-6 production in myoblasts (A) and myotubes, the terminally differentiated and functional muscle cells
(B). The ELISA assay showed significant reduced levels of IL-6 after 30 min IL-1β (10 ng/ml) coincubation with canakinumab (10 nM) in both cell types (p < 0.0001).