Fig 2: CYP27A1 overexpression does not affect migration and invasion in glioblastoma U87MG and LN-229 cells. (A) Matrigel invasion and Transwell migration assays for U87MG cells transfected with CYP27A1-expressing plasmids or the empty vector (mock). n=3. (B) Matrigel invasion and Transwell migration assays for LN-229 cells transfected with CYP27A1-expressing plasmids or the empty vector (mock). n=3. Error bars indicate standard error of the mean. CYP27A1, cytochrome P450, family 27, subfamily A, polypeptide 1.

Fig 3: Kaplan–Meier graphs showing the joint prognostic impact of CYP27A1 expression with NHG and Ki67.a CYP27A1/NHG, b CYP27A1/Ki67.

Fig 4: CYP27A1 promotes proliferation in glioblastoma U87MG cells. (A) Western blot analysis for CYP27A1 in U87MG cells. U87MG cells were transfected with CYP27A1-expressing plasmids or the empty vector (mock). ß-actin was used as the loading control. n=3. (B) MTT assay for U87MG cells. U87MG cells were transfected with CYP27A1-expressing plasmids or the empty vector (mock) and cellular viability was then measured at the indicated time points using an MTT assay. n=3. (C) BrdU incorporation assay for U87MG cells. Representative micrographs and quantification of BrdU incorporating-cells following transfection with CYP27A1-expressing plasmids or the empty vector (mock). n=3. (D) Western blot analysis for c-myc, RB, Ki67, CDK2, p21, p53, PDCD4 and SOX2 in U87MG cells transfected with CYP27A1-expressing plasmids or the empty vector (mock). ß-actin was used as the loading control. n=3. Error bars indicate standard error of the mean. CYP27A1, cytochrome P450, family 27, subfamily A, polypeptide 1; BrdU, bromodeoxyuridine; RB, retinoblastoma; CDK2, cyclin-dependent kinase 2; PDCD4, programmed cell death protein 4; SOX2, sex determining region Y-box 2.

Fig 5: Effect of HCQ in the presence and absence of IFN? on the expression of CYP27A1. Western blot analysis using CYP27A1 specific antibody after 18 h incubation of THP-1 macrophages. ß-actin was detected on the same membrane as a loading control. Top: Immunoblot results presented as a graph for CYP27A1 protein expression with band densities normalized against ß-actin. Bottom: Immunoblot with CYP27A1 and ß-actin detected as immunoreactive bands. The data represent the mean and SEM of three independent experiments (n = 3). Western blot and densitometry analyses showed that CYP27A1 expression decreased with HCQ in the presence of IFN?. Light green bars = media, no IFN?; Dark green bars = IFN?, 100 units/mL added to media. * p < 0.05 for the interaction effect only between IFN? and HCQ.