Gibco opti mem glutamax reduced serum media

Lenti-X cells were transfected in Gibco opti-MEM Glutamax reduced serum media (Thermo Fisher Scientific) with the pCDH-EF1-HA-FGFR2b-T2A-mApple or pCDH-EF1-HA-FGFR2b-APEX2-T2A-mApple viral vectors, alongside VSV-G envelope expressing plasmid pMD2.G and lentiviral packaging plasmids pMDLg/pRRE and pRSV-Rev (all generously gifted from Dr. Hurlstone), using FuGENE® HD Transfection Reagent (Promega), following manufacturer instructions. After 48 h, the lentivirus-containing media was sterile filtered using a 0.22 μm syringe filter and stored at −80 °C. The lentiviral media was added to HeLa or T47D_FGFR2b^KO in Gibco opti-MEM Glutamax reduced serum media (Thermo Fisher Scientific) containing 10 ng/mL Polybrene Infection/Transfection Reagent (gifted from Dr. Hurlstone). Colonies were selected and protein expression was then confirmed by Western blot. These cell lines are referred to as HeLa_FGFR2b^ST, HeLa_FGFR2b-APEX2^ST and T47D_FGFR2b^KO_FGFR2b-APEX2^ST.

All transfections were carried out in Gibco opti‐MEM glutamax reduced serum media (Thermo Fisher Scientific). For RNA interference, all cells were transfected using Lipofectamine RNAiMax (Thermo Fisher Scientific), according to manufacturer instructions. Validated double‐stranded stealth siRNA oligonucleotides were used for RNA interference. siRNA Universal Negative Control #2 (Sigma‐Aldrich) was used as a control in all RNA interference experiments. BT549 and BT20 cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions, 24 h after RNA interference transfection where indicated. T47D cells were transfected using Escort IV according to manufacturer instructions, same as above. Assays were performed 36 h after transfection, as previously described (Francavilla et␣al,2016 (link)). Where assays were performed more than 36 h after transfection, RNAi and expression were assessed at time of assay to confirm expression.

All transfections were carried out in Gibco opti-MEM glutamax reduced serum media (Thermo Fisher Scientific). HeLa cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions, 24 h after RNA interference transfection where indicated. T47D and BT20 cells were transfected using Escort IV according to manufacturer instructions, same as above. Assays were performed 36 h after transfection. A mock control was used if unspecified.
HeLa cells stably expressing HA-FGFR2b or HA-FGFR2b-APEX2 are referred to as follows: HeLa_FGFR2b^ST, HeLa_FGFR2b-APEX2^ST. Cells were transiently transfected with the following constructs: eGFP (GFP in text), dynamin_K44A-eGFP (DnDNM2 in text), eGFP-RAB11_S25N (DnRAB11 in text), eGFP-RAB11 (wtRAB11 in text), eGFP-RAB11-APEX2 (RAB11-APEX2 in text) and eGFP-APEX2 (GFP-APEX2 in text).