Fig. 6
TFE3 and TFEB increase TMEM55B expression to cluster lysosomes during starvation. a–c ARPE-19 cells were infected with adenovirus expressing TFE3-S321A-Myc, TFEB-S211A-FLAG, or null virus for 36 h. 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, n = 4). b Representative immunoblot of lysates from TFE3-S321A-Myc and TFEB-S211A-FLAG expressing cells. c Quantification of TMEM55B protein levels. d Control MEFs untreated or starved in EBSS for 4 h. Cells were fixed, permeabilized, and immunostained with antibodies against TFE3. e–g Relative quantitative real-time PCR analysis of e MCOLN1, f TMEM55B and g TMEM55A 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) n = 6 from three independent experiments. h HeLa cells stably expressing control or TFEB shRNAs were starved in EBSS for 4 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. i HeLa cells expressing control, TMEM55B, and JIP4 RNAis were starved in EBSS for 4 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1. j Quantification of lysosome distribution, percentage of total fluorescence signal detected >15 μm from nuclear rim. Quantified results are presented as mean ± s.e.m. using two-tailed t-test ∗ P < 0.05, ∗∗ P < 0.005 were considered significant, n ≥ 30. k Immunoblot of lysates from HeLa cells treated with siControl or siJIP4 siRNA, then starved in EBSS for 4 h, or starved in EBSS for 4 h with 2 h 100 nM bafilomycin. Note that all the samples were run in the same gel but are presented in two panels due to space limitations. l quantification of LC3II/Actin ratios from siControl or siJIP4-treated HeLa cells starved in EBSS 4 h with 100 nM bafilomycin for 2 h. Quantified results are fold increase of LC3II/Actin from siControl after starvation and bafilomycin treatment and data are presented as mean ± s.e.m. using two-tailed t-test **P < 0.005, n = 3. m HeLa cells treated with siControl or siJIP4 siRNA and starved with EBSS for 4 h and 100 nM bafilomycin for 2 h. Cells were fixed, permeabilized, and immunostained with antibodies against LAMP-1, LC3, and DAPI. Scale bars, 20 μm
