(A) Chemical structures of cholesterol and cholestenone showing A–D rings (red text). The 3-hydroxyl group of cholesterol is dehydrogenated to a keto moiety in the A ring in cholestenone (highlighted in green). (B) Cholestenone was quantified from BMDMs infected with M. tuberculosis at MOI 1, 5, and 10, at 3, 24, 72, and 120 hpi. Uninfected (UI) cells or those infected with heat-killed M. tuberculosis (HK) were used as controls. Plot shows average of 2 independent experiments.*P = 0.03, **P = 0.007 for 120 hours, calculated using Mann-Whitney test. (C) Cholestenone levels in PMA-differentiated THP-1 macrophages that were uninfected or M. tuberculosis–infected at MOI 10, 72 hpi. (D) Growth of M. tuberculosis was compared in minimal medium supplemented with either a vehicle control, 100 μg/mL 25-HC, or 100 μg/mL cholesterol. Plot shows values from 1 experiment representative of 3 independent experiments. (E) Cholestenone abundance and (F) corresponding M. tuberculosis CFU in IFN-γ–activated and naive BMDMs that were uninfected or M. tuberculosis–infected at MOI 10 at the indicated time points. (G and H) The direct effect of cholestenone on M. tuberculosis growth was assessed in culture medium using absorbance measurements (G) and CFU (H). (C, E, and F) Plots show mean ± SEM from at least 3 independent experiments. **P = 0.007, ****P < 0.0001 calculated using Student’s t test (C), and 1-way ANOVA with Tukey’s multiple comparisons test (E and F). (G and H) Plots show average of 2 independent experiments. Also see Supplemental Figure 6. (C and E) The dotted line on the y axis represents the limit of detection accuracy as determined by the standard curve. For all macrophage experiments, one million cells were infected and the samples were extracted in 500 μL 80 % methanol solution containing 0.1 μg internal standard. The methods used for calculating cholestenone concentrations are detailed in the Supplemental Material.