Cells were treated with recombinant adenovirus expressing Cre recombinase (Ad-Cre) or empty Ad (titer 1.3×1011; Vector Core, University of Michigan, Ann Arbor), dissolved in complete media at MOI=200 and a minimal volume covering the cells. Four h later, complete media was added to a volume optimal for the dish size. Media was replaced 14 h later with fresh complete media. Pikfyve KO MEFs (100 mm confluent dishes) were transfected on day 4 post Ad-Cre treatment by electroporation using 50 μg of pEGFP-2xFYVEPIKfyve (14 (link)) or pTag-YFP-Vps34wt cDNAs under a single pulse of 975 μF and 0.16 kV (Biorad-Gene pulser II) as described previously (36 (link)). Vps34 KO podocytes were transfected with the above cDNAs by Lipofectamine 3000 transfection kit (Invitrogen) following manufacturer protocols. Where indicated, 24 h post transfection, Vps34 KO podocytes were treated (60 min; 37°C) with 1 μM of the YM201636 inhibitor (Symansis NZ) dissolved in DMSO or only with vehicle.
Recombinant adenoviruses expressing HA-PIKfyvewt and GFP from separate CMV promoters (AdGFP-PIKfyvewt) or only GFP (AdGFP) were used at MOI=10, with MOI=1, being the lowest dilution reaching 100% infection efficiency in HEK293 cells for 14 h (9 (link)). The chosen MOI delivered infection in ~20–25% of the total Pikfyve KO or Vps34 KO cells, the non-infected cells on the same dish thereby serving as controls.
Recombinant adenoviruses expressing HA-PIKfyvewt and GFP from separate CMV promoters (AdGFP-PIKfyvewt) or only GFP (AdGFP) were used at MOI=10, with MOI=1, being the lowest dilution reaching 100% infection efficiency in HEK293 cells for 14 h (9 (link)). The chosen MOI delivered infection in ~20–25% of the total Pikfyve KO or Vps34 KO cells, the non-infected cells on the same dish thereby serving as controls.