ssRNA40- and CL097-induced autophagy in macrophages is mediated through CYP27B1 and the VDR.

<p>(A–B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) <i>Top</i>, qRT-PCR for CYP27B1 after 6 h. <i>Bottom</i>, immunoblots of CYP27B1 using antibody to CYP27B1 or <i>β</i>-actin. (B) <i>Top</i>, qRT-PCR for VDR after 6 h. <i>Bottom</i>, immunoblots of VDR using antibody to VDR or <i>β</i>-actin. (C–D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) <i>Left</i>, immunoblots performed with antibodies to CYP27B1, VDR and <i>β</i>-actin. <i>Right</i>, immunoblots of LC3B isoforms using antibody to LC3B or <i>β</i>-actin. (D) Cells were fixed and permeabilized then stained with antibody to LC3B. Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s.e.m., <i>n</i> = 4. (E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or <i>β</i>-actin. Bar charts are reported as mean ± s.e.m., <i>n</i> = 4. * <i>p</i><0.05.