Fig. 3. RPS27L knockdown destabilizes FANCD2 and FANCI via p62-mediated autophagy-lysosome pathway.

A, B Silencing of RPS27L shortens the protein half-lives of FANCD2 and FANCI protein. H1299 (A) and A549 (B) cells were infected with indicated lentivirus for 48 h, followed by CHX treatment for indicated periods of time before harvesting for IB with indicated Abs (left). Densitometry quantification was performed with ImageJ, and the decay curves are shown (right). LE: longer exposure; SE: shorter exposure. * Non-specific band. C, D Chloroquine (CQ), but not MG132, abrogates FANCD2 and FANCI reduction induced by RPS27L knockdown. H1299 cells (C) and A549 cells (D) were infected with indicated lentivirus, and then harvested for IB after treatment with MG132 (1 µM) for 12 h or CQ (50 µM) for 24 h. E Beclin 1 knockdown abrogates FANCD2/FANCI reduction upon RPS27L knockdown. H1299 cells were transfected with indicated siRNA oligos, and then harvested for IB. F FANCD2/FANCI binds to p62. 293 cells were transfected with indicated plasmids for 48 h, and then IP with FLAG beads before harvesting for IB with indicated Abs. WCE: whole-cell extract. G RPS27L knockdown promotes FANCD2/FANCI binding to p62. H1299 cells were transfected with indicated siRNA oligos. After 24 h, cells were transfected with mock vector or FLAG-p62 for additional 48 h, and then treated with CQ (50 µM) for 24 h before harvesting for IP with FLAG beads, followed by IB with indicated Abs. The upper band of FANCD2/FANCI is the monoubiquitylated form, and the lower band is the nonubiquitylated form.