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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2020 Jan 30;40(3):714–732. doi: 10.1161/ATVBAHA.119.313832

Figure 4. Statin treatment of BMDMs led to an Rac1 isoprenylation-dependent increase in IL-1β expression.

Figure 4.

(A) ApoE−/− BMDMs were pretreated for 24 hours with or without atorvastatin (10 μM) and then primed with or without LPS (10 ng/ml) followed by exposure to cholesterol crystals (1000 μg/mL) or vehicle control for another 24 hours, and cell lysates underwent real-time PCR quantification of IL-1β mRNA (***, P<0.0001 ANOVA; n=6 biological replicates from 6 mice; 3 male and 3 female). (B) ApoE−/− BMDMs were treated as in (A) followed by by ELISA on culture supernatants for IL-1β (***, P<0.0001 ANOVA; n=6 biological replicates from 6 mice; 3 male and 3 female). (C) ApoE−/− BMDMs were treated as in (A) followed by real-time PCR quantification of TNF-α mRNA from cell lysates. (D) ApoE−/− BMDMs were treated as in (A) followed by ELISA on culture supernatants for TNF-α. (E) Resting or LPS-primed (10 ng/ml) ApoE−/− BMDMs were treated with or without 1000 μg/mL cholesterol crystals for 24 hours in the setting of indicated atorvastatin concentrations followed by ELISA on culture supernatants for IL-1β (**, P<0.0001 ANOVA; n=6 biological replicates from 6 mice; 3 male and 3 female). (F) Relative luciferase activity in lysates from BMDMs transfected with a NF-κB responsive luciferase construct and the treated with vehicle control (DMSO) or atorvastatin (10 μM) for 24 hours (*, P<0.05 t-test; n=6 biological replicates from 6 mice; 3 male and 3 female). (G) Relative luminescence as a measure of ROS production in BMDMs treated with vehicle control (DMSO) or atorvastatin (10 μM) for 24 hours (***, P<0.0001 t-test; n=6 biological replicates from 6 mice; 3 male and 3 female).

(I) BMDMs from ApoE−/−CSF1RmcmRac1fl/fl mice were treated with vehicle control or atorvastatin (10 μM) with or without either FPP (5 μM), GGPP (5 μM), or squalene (5 μM) supplementation for 24 hours with or without 4-hydroxytamoxifen (4-OHT) to induce Rac1 deletion and then were primed with or without LPS (10 ng/ml) and exposed to cholesterol crystals (1000 μg/mL) or vehicle control for 24 hours followed by ELISA on culture supernatants for IL-1β (**, P=0.0001; n=6 biological replicates from 3 mice; 2 male and 1 female). Quantitative data are displayed as mean ± SD.