Extended Data Fig. 7: BC1618 is anti-inflammatory in vitro.
From: A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance
a, THP1 cells stably expressing an NF-κB inducible luciferase (secreted) reporter were treated with LPS (100 ng/ml) and BC1618 at indicated concentrations for 6 h or 24 h before supernatant was collected for luciferase activity assay following the manufacturer’s protocol. Data are shown as the mean ± SEM of three independent biological replicates, and significance was measured by one-way ANOVA with Dunnett’s test of multiple comparisons relative to 0µM BC1618+LPS. For 6hs, 0.0032µM: p=0.0868; for 0.016,0.08, 0.4, 2, and 10µM: p<0.0001. For 24hr, 0.0032µM: p=0.0002; for 0.016,0.08, 0.4, 2, and 10µM: p<0.0001. b, 50K PBMC cells were cultured in 96 well plates before being exposed to BC1618 at indicated concentrations for 18 h. Cells were then treated with LPS (10 ng/ml) for an additional 4 h. Media were then collected and TNF and IL-1 concentration were determined by ELISA. Data are shown as the mean ± SEM of three independent biological replicates, and significance was measured by one-way ANOVA with Dunnett’s test of multiple comparisons relative to 0µM BC1618 for both cytokines. For TNF treatment, 0.008µM: p=0.2212; 0.04µM: p=0.0375; 0.2µM: p=0.3585; for 1, 5, and 25µM: p<0.0001. For IL-1 treatment, 0.008µM: p=0.7497; 0.04µM: p=0.0919; 0.2µM: p=0.0126; 1µM: p=0.0028; 5µM: p=0.0015; 25µM: p=0.0065. Significance is also indicated as follows: *, P <0.05; **, P < 0.01; ***, P <0.001; ****, P<0.0001 (c) PBMC cells (1 mL at 1.0 x 106/ml) were treated with BC1618 (5 µM) for 18 h. Cells were then treated with 100 ng/ml LPS for additional 4 h. Cytokine release was monitored by the human cytokine array (R&D systems). The results from a cytokine array dot blot are shown and quantitated (Data are shown as the mean of two independent biological replicates).
