Generation of stable cell lines by viral transduction

To generate pBMN-HA-vector, pBMN-HA-Rab7^WT, pBMN-HA-Rab7^Q67L, and pBMN-HA-Rab7^T22N constructs, the pBMN-Z retroviral vector (Gary Nolan, Stanford University, Addgene # 1734) was digested with Sal1/BamH1 before cloning using the HiFi DNA Assembly 1232 Kit (New England Biolabs, Cat # E5520S) according to the manufacturer’s instructions. pBMN-HA vector, pBMN-HA-Rab7^WT, pBMN-HA-Rab7^Q67L, and pBMN-HA-Rab7^T22N were generated by PCR amplification of pCGN-HA⁶², or pMH-SFB-Rab7^WT, pMH-SFB-Rab7^Q67L, or pMH-SFB-Rab7^T22N (Supplementary Table ¹), which were a kind gift from Subbareddy Maddika⁶³. pLVX-TRE3G-mCherry-Myc-INPP4B^WT and pLVX-TRE3G-mCherry-Myc-INPP4B^C842A lentiviral plasmids were generated by digesting pLVX-TRE3G-mCherry (Clontech, Cat # 631352) with Nde1/Mlu1. Myc-INPP4B^WT or Myc-INPP4B^C842A19 were amplified with with Nde1/Mlu1 restriction sites, then digested and cloned into the Nde1/Mlu1 site of pLVX-TRE3G-mCherry. All plasmid DNA sequences were verified by Sanger sequencing (Micromon, Monash University, Australia).

Retroviral transductions were carried out as previously described⁶⁴. For all lentiviral transductions, cells were seeded into a 12-well plate at a density of 4.25 × 10⁴ cells per well. The next day, fresh media containing 10 μg/mL polybrene and lentiviral or retroviral particles at a multiplicity of infection of 1 was added. Cells were centrifuged at 230 × g for 1 h at room temperature then incubated at 37 °C. After 24 h, the culture media was aspirated and cells were washed three times in PBS to remove virus particles before fresh media was added. Cells transduced with lentiviral particles encoding pHIV-1SDmCMV.pre GFP-vector or pHIV-1SDmCMV.pre GFP-INPP4B⁶ were selected by fluorescent activated cell sorting (FACS) (Flowcore, Monash University, Australia). Cells transduced with lentiviral particles encoding pLKO.1-puro Non-Mammalian shRNA (Sigma, Cat # SHC002), INPP4B #1 mission^® shRNA (Sigma, Cat # TRCN0000052721), INPP4B #2 mission^® shRNA (Sigma, Cat # TRCN0000052722), HGS mission^® shRNA (Sigma, Cat # TRCN000038-0920), PIK3C2A #1 mission^® shRNA (Sigma, Cat # TRCN0000002228), or PIK3C2A #2 mission^® shRNA (Sigma, Cat # TRCN0000002229) lentiviral particles were selected by culturing cells in media containing 1 μg/mL puromycin (Sigma, Cat # P9620) for 1 week, then maintained in media containing 0.5 μg/mL puromycin. Cells co-transduced with lentiviral particles encoding pLVX-EF1α-Tet3G and pLVX-TRE3G-mCherry, pLVX-TRE3G-mCherry-Myc-INPP4B^WT or pLVX-TRE3G-mCherry-Myc-INPP4B^C842A were selected by culturing in media containing 1 μg/mL puromycin and 600 μg/mL G418 (AG Scientific, Cat # G-1033) for 1 week, then maintained in media containing 0.5 μg/mL puromycin and 300 μg/mL G418. Cells were plated into antibiotic-free media for all experiments. To induce expression of Myc-INPP4B^WT or Myc-INPP4B^C842A, 10 µg/mL doxycycline was added to media when cells were plated.