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. 2014 Nov 13;16(1):87–96. doi: 10.15252/embr.201439218

Figure 4. EMP1 is required for bronchial epithelial tight junction formation and function.

Figure 4

  1. 16HBE cells were stably infected with lentiviral vector pLKO alone or expressing EMP1 shRNAs (1 or 2). Total RNA was isolated and analysed for EMP1 expression using TaqMan/qPCR with a GAPDH control. Wild-type cells treated with 500 nM GSK1120212 or siSOS1 for 4 days were also analysed. Error bars denote mean ± SEM, and dots indicate individual data points. ***P < 0.0002 (GSK = 0.0002, siSOS1 = 0.0001); ****P < 0.0001.
  2. Cells as in (A) were fixed and stained for ZO-1, E-cadherin and DNA. Scale bar, 20 μm.
  3. Quantification of tight junction phenotype of cells as in (A). > 500 cells were counted per sample/experiment, across n = 3 independent experiments (dots indicate individual data points). Error bars denote mean ± SEM. *P = 0.0148; **P = 0.006.
  4. Transepithelial resistance (TER) was measured in 16HBE cells as in (A) on day 4 post-seeding. Error bars denote mean ± SEM, and dots indicate individual data points. **P = 0.0036; ***P = 0.0005.
  5. Cells as in (A) were seeded on glass-bottomed dishes for 4 days. FM 4–64 dye was applied to the media and confocal z-stacks acquired.
  6. 16HBE cells were fixed and stained for endogenous EMP1 and DNA using two different commercial antibodies. Scale bar, 20 μm.
  7. 16HBE cells were seeded on glass coverslips for 10 days to form a mature, polarised monolayer. Cells were fixed and costained for EMP1 and ZO-1 and then analysed by confocal microscopy. A representative z-stack is presented; arrows indicate ZO-1-positive tight junctions.

Data information: All data are representative of n = 3 independent experiments.