Fig. 7

TFEB/3 and SREBF2 co-operate to regulate TMEM55B levels in response to changes in cholesterol levels. a, b HeLa cells were treated with drugs to deplete cellular cholesterol for 120 h, treated with 10 μM U18666A for 18 h, or left untreated. a Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH mRNA, using two-tailed t-test **P < 0.005 were considered significant) n = 3. b Representative immunoblot of lysates from cholesterol depleted cells. c Control MEFs were untreated or incubated in 10 μM U18666A for 18 h. Cells were fixed, permeabilized, and immunostained with antibodies against TFE3. d Relative quantitative real-time PCR analysis of TMEM55B mRNA transcript levels from null- or TFE3/TFEB knockout MEFs, untreated or starved in EBSS for 4 h (mean ± s.e.m. of the RNA fold change of indicated TMEM55B normalized to GAPDH, using two-tailed t-test *P < 0.05 were considered significant) n = 6 from three independent experiments. e HeLa cells depleted of TMEM55B, JIP4, or control RNAi were starved in EBSS for 4 h and then immunostained with antibody against LAMP-1. f Control MEFs treated with control and SREBF2 siRNAs and TFEB/TFE3 KO MEFs treated with SREBF2 siRNAs were incubated with 10 μM U18666A for 18 h and the TMEM55B mRNA transcript levels were quantified by qRT-PCR (mean ± s.e.m. of the RNA fold change normalized to TMEM55B levels in siControl-treated cells, using two-tailed t-test *P < 0.05, **P < 0.005 were considered significant) n = 6 from three independent experiments. g Immunoblot of lysates from primary skin fibroblasts from Niemann-Pick C (NPC1) patient 1, NPC1 patient 2, and genetic matched control patient. h NPC1 patient fibroblasts depleted of JIP4 with RNAi. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1, JIP4, and DAPI. i Model of TMEM55B transcriptional regulation. Nutrient deprivation inactivates mTOR to allow TFEB/3 translocation to the nucleus to activate transcription of TMEM55B and induce lysosome retrograde trafficking. Decreased sterol levels at the ER stimulates SREBF2 to translocate to the nucleus to activate transcription of TMEM55B and induces lysosome retrograde trafficking. TMEM55B recruits scaffold JIP4 that binds dynein–dynactin motor complex. Scale bars, 20 μm