Figure 6.
Analysis of the effects of LXR agonist TO901317 on LXRα target gene regulation in infected cells. (A) Pictures of representative results of CFU analysis of DMSO- or TO901317- (20 nM) treated THP-1 cells infected with H37Ra for 5 days: dilution 10−5 of H37Ra spread on 7H10 plates and incubated for 3 weeks. (B) Quantification of CFU assay results at day 0 and day 5 after infection and treatment, n = 3. (C) Assessment of Mtb replication after DNA and RNA extraction was performed by (RT-)qPCR analysis of the ratio of 16SRNA mycobacterial RNA to genomic DNA. (D) Results of IPA (Ingenuity®) pathway enrichment analysis for genes regulated at least 2-fold by treatment with LXR agonist TO901317. Green is for downregulated genes, and red is for upregulated genes. (E) RT-qPCR analysis of expression of genes encoding cholesterol transporters ABCA1, ABCG1, ABCG4 and ABCG5 in uninfected (Ni) or H37Ra (Ra)-infected THP-1 cells treated 24 h with DMSO- or TO901317 (20nM). (F) Imaging of Lipid Droplets (LD) in infected (H37Ra) vs uninfected (NI) THP-1 cells treated with DMSO (veh.) or TO901317, and stained with Bodipy 495/503 and DAPI (Axiovert microscope, X100 objective). (G) Quantification of LD, by size and intensity, number of LD per cell and the total lipid droplet content in H37Ra-infected THP-1 cells treated with DMSO or TO901317. Measurement was done on 20 different images for each condition and on at least 10 cells per image. Measurement and quantification were done using Imaris. **P < 0.05, *P < 0.005. (H) Cholesterol efflux inhibitors Ritonavir (30μM) or Nelfinavir (10 μM) reverse the antimycobacterial effects of TO901317. Quantification of Mtb replication after DNA and RNA extraction was performed by qPCR. Results are represented as expression of 16SRNA normalized to genomic DNA.