Fig. 3. SLFN5 TP sites are essential for U2OS cell proliferation and viability and required for B55α binding.

A Model of SLFN5 regulation and binding to PP2A-B55α. SLFN5 minimal TP consensus sequences can be dephosphorylated by PP2A-B55α (upper panel). Serine-to-Threonine substitution reduces PP2A-B55α binding to SLFN5 (middle panel). A non-phosphorylatable SLFN5 version where threonine residues have been replaced by alanine prevents PP2A-B55α binding and SLFN5 activity (lower panel). B, C Cell viability and cell growth are impaired in SLFN5-depleted U2OS cells and can be rescued by expression of RNAi resistant WT SLFN5. Cells were treated for three days with RNAi against SLFN5 or Luciferase and grown for further 9 days in presence or absence of Doxycycline. Cell viability was assessed by SRB assay (B), or cell confluence was monitored over 6 days under the same conditions by Incucyte growth assay to assess cell proliferation (C). The graphs statistically illustrate the results from three independent experiments (B, C). D, E B55α interacts with the SLFN5 DNA binding domain (DBD). WT, SP, and AP variants of V5-tagged full length SLFN5 and its single domains were transiently transfected into inducible YFP-B55α-HeLa cells. YFP-B55α expression was induced by addition of doxycycline. Immunoprecipitation was performed using an antibody to GFP. SLFN5 recovery was probed by Western blotting using a V5-antibody. The Western blot shows a representative experiment from three independent experiments (E). F Depletion of PP2A catalytic subunits α and β (siPPP2C) in inducible YFP-B55α-HeLa cells impairs WT SLFN5 recovery. Cells were treated with RNAi oligomers targeting both isoforms of PPP2C for 48 h and WT SLFN5 recovery was assessed by Western blotting using V5-antibody after pulldown of the YFP-tagged B55α regulatory subunit of PP2A. YFP-B55α expression was induced by treatment with Doxycycline. The Western blot shows a representative experiment from three independent experiments.