Inpp4b Regulation of Autophagy and Lysosomal Dynamics

Immortalized Inpp4b^+/+ and Inpp4b^−/− MEF were generated as previously detailed (51 (link)). MEF and U2OS cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). MEF or U2OS cells were transiently transfected with pTWIST-mCherry, pTWIST-Inpp4b-mCherry, mCherry-Lamp1, mCherry-EGFP-LC3B, pEGFP, GFP-Inpp4b, GFP-Inpp4b (C845A), and pEGFP-TFEB. U2OS cells were stably transfected with mCherry-Lamp1 through selection with 200 μg/ml G418 for 10 days. Transfections of MEF and U2OS cells performed with Fugene HD (Promega) at 3:1 of DNA:Fugene ratio for 24 h followed by washing and supplementation with complete DMEM growth media. siRNA-mediated gene silencing for INPP4B in U2OS cells carried out using DharmaFECT1 Transfection reagent (GE Dharmacon). Briefly, 0.1 nmol of nontargeting or Inpp4b siRNA (GE Dharmacon) mixed with 2 μl of DharmaFECT1 Transfection reagent in DMEM media without FBS was added to U2OS cells for 24 h, followed by washing off the transfection mix with PBS and growth of cells for 48 h with treatment before imaging and Western blot. MEF and U2OS cells treated with apilimod or VPS34-IN1 (Selleck Chemicals) to inhibit PIKfyve or VPS34 functions respectively at doses and durations indicated.