Plat e cells

Retrovirus packaging was described previously
[13 (link)]. In brief, to isolate the retrovirus, pMX-IRES-GFP (the control retrovirus vector encoding GFP, green fluorescent protein) and pMX containing constitutively active (CA)-NFATc1 were transiently transfected into Plat-E cells (platinum-E retrovirus packaging cell line; Cell Biolabs, Inc.) with Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. Viral supernatant was collected from the culture media 48 h after transfection. BMMs were incubated with viral supernatant in the presence of polybrene (10 μg/ml) for 8 h. The infection efficiency of the retrovirus was determined by GFP expression and was always greater than 80%. After infection, BMMs were induced to differentiate in the presence of M-CSF (30 ng/ml) and RANKL (10 ng/ml) for 4 days.

Immortal melanocyte cell lines were used in this study – wild-type melan-Ink4a-Arf-1 (from C57BL/6J, a/a, Ink4a-Arf^−/− mice, formerly called melan-Ink4a-1, referred to here as WT or melan-Ink4a) (Ha et al., 2007 (link)), and AP-3-deficient melan-mh1 [from C57BL/6J Ap3d^mh/mh, referred to here as AP-3⁻ (melan-mh)] or melan-pe1 [from C57BL/6J Ap3b1^pe/pe, referred to here as AP-3⁻ (melan-pe)] (Theos et al., 2005 (link)). Cells were maintained as described previously (Sviderskaya et al., 2002 (link)).
Plasmids were transfected into the melanocytes or PLAT-E cells (Cell Biolabs) by Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Melanocytes were also transduced with retroviruses (containing different pBMN-IRES-Hygro or pRSshRNA plasmids) isolated from PLAT-E cells (Morita et al., 2000 (link)). Post transduction, melanocytes were selected twice with hygromycin (200 μg/ml) or puromycin (2 μg/ml) on the 2nd and 4th day. In some experiments, shRNA knockdown cells were transfected with GFP–STX13 or GFP–VAMP7 using Lipofectamine 2000 reagent. Stable wild-type (melan-Ink4a) melanocytes expressing different constructs of Myc–STX13 were generated by the retroviral transduction method and used for immunoelectron microscopy and VAMP7 knockdown in Fig. 6A–D. STX13-knockdown cells were rescued by transfecting the cells with GFP–hSTX13^WT.

HepG2, Hep3B, HLE, HLF, HuCCT1, and HuH28 cells were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). Plat-E cells were obtained from Cell Biolabs Inc. (San Diego, CA). HeLa, NIH/3T3, TALDO1 KO, HepG2, Hep3B, HCE, and HCF cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO) supplemented with 10% foetal bovine serum (FBS). Plat-E cells were cultured in DMEM containing 10% FBS, puromycin (1 μg/mL), and blasticidin S (10 μg/mL). HuCCT1 and HuH28 were cultured in RPMI-1640 (Sigma-Aldrich) supplemented with 10% FBS. The cells were maintained in a 37 °C incubator with 10% CO₂-humidified air.