Fig. 7

TANK overexpression impairs the protective effect of PLK1 on LPS-induced mitochondrial dysfunction, apoptosis and high permeability in Caco-2 cells. Caco-2 cells were transiently transfected with pCDNA3.1-PLK1 (a PLK1 knock-in plasmid) or/and pCDNA3.1-TANK (a TANK knock-in plasmid) for 24 h and then treated with LPS (50 μg/mL) for 24 h. A The location of NF-κB was examined after the indicated treatments. NF-κB is stained green; the nuclei are stained blue. The scale bar represents 20 μm. B Representative Western blots showing the indicated proteins in the NF-κB pathway, and the lower graph shows the relative band densities. C Morphological alterations in mitochondria were examined by TEM (scale bar = 0.5 μm). Yellow arrows indicate the relatively normal mitochondrial shape in the sham group. Red arrows denote deformed mitochondria with loss of clearly defined cristae. Green arrows indicate the rescued mitochondrial shape. Blue arrows indicate the worsened damage to mitochondrial shape. D The relative fluorescence intensity of ROS in the Caco-2 cells. E The ratio of JC-1 red/green fluorescence intensity which reflecting the MMP level was shown in graph. F Representative Western blots showing mitochondrial fusion (Mfn1, Mfn2, OPA1) and fission (Drp1 and Fis1) markers on the left, and the graph on the right shows the relative band densities. The data in the graphs are expressed as the mean ± SD. G Apoptosis was analysed by Annexin V-FITC/7-AAD double-labelling assays. The graph shows the proportion of apoptotic cells. H The levels of apoptotic markers were measured by Western blots. The graph shows the relative band densities. I The levels of ZO-1 and Occludin were analysed by Western blots. The graph shows the relative band densities. J TEER levels in each group. The data in the graphs are expressed as the mean ± SD. Each result was replicated in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001