Figure 6. miR-21 silencing of PTEN inhibited GSK3β that in turn inhibited inducible NFκB activation.
A, Phosphorylation of GSK3β (Serine 9) in THP-1 monocytic cells where stable knock down of miR-21 was achieved following lentiviral transduction with lenti-miR-000-zip or lenti-miR-21-zip vectors followed by LPS treatment (1ug/ml). Data are mean ± SD (n=3). *, p<0.05 compared to control lenti-miR-000-zip cells. B, Phosphorylation of GSK3β protein was measured using Western blot in MDM infected with adenovirus-PTEN or adenovirus GFP vectors followed by LPS treatment. Data are mean ± SD (n=3). *, p<0.05 compared to control adenovirus GFP infected cells. C, Phosphorylation of NF-κB p65 protein was measured using Western blot in MDM treated with 10 μM SB 216763 (GSK3β inhibitor) followed by LPS treatment. Quantification was performed using densitometry. Data are mean ± SD (n=3). *, p<0.05 compared to control cells. D, Phosphorylation of IκB-α protein in MDM treated with 10 μM SB 216763, followed by LPS treatment. Data are mean ± SD (n=3). *, p<0.05 compared to control cells. E, Phosphorylation of IKKβ protein (Serine 181) in MDM treated with 10 μM SB 216763, followed by LPS treatment. Data are mean ± SD (n=3). *, p<0.05 compared to control cells. F, TNFα protein expression was measured in MDM treated with 10 μM SB 216763 followed by LPS treatment. Data are mean ± SD (n=4). *, p<0.05 compared to LPS treated cells; §, p<0.05 compared to SB 216763 treated group. G, TNFα protein expression in MDM pretreated with 10μM SB216763 followed by efferocytosis and LPS treatment. Data are mean ± SD (n=3). *, p<0.05 compared to Effrlo group; §, p<0.05 compared to SB 216763 non-treated group.