Figure 2.

Effects of Tid1-S overexpression or knockdown on the translocation of EGFR into mitochondria. (a) Schematic illustration of wild-type Tid1-S-HA (Tid1-S-Wt) and the DnaJ domain mutant Tid1-S-HA (Tid1-S-Mut), in which the amino acid sequence H121P122D123 (wild-type) was replaced with Q121N122A123 (mutant) in the DnaJ domain of Tid1-S. (b) CL1-5 cells were transfected with Tid1-S-Wt, Tid1-S-Mut, or empty vector (control) and cultured for 24 h. Representative IF staining with anti-HA, -EGFR and -MTCOI antibodies is shown in the left panel. The colocalization of EGFR and MTCO1 in mitochondria could be visualized as yellow-color in the EGFR/MTCO1 Merge. The colocalization of HA, EGFR, and MTCOI in mitochondria was detected as white color in the HA/EGFR/MTCO1 Merge. The expression of HA-tagged Tid1-S-Wt or Tid1-S-Mut in the transfected CL1-5 cells was determined by Western blotting, and is shown in the right-upper panel. The arrows indicated by ‘up-S’ and ‘p-S’ show the positions of the unprocessed form (up) and the processed form (p) of Tid1-S-HA (S), respectively. β-Actin was used as a loading control. The colocalization of HA-Tid1-S, EGFR and MTCOI in~200 cells was analyzed using the MetaMorph software. The scores are expressed as a fold-increase over that of the vector-transfected control cells, which was set as 1. The data shown in the right-lower panel were from three independent experiments. (c) A549 cells were transfected with siTid1 or siN for 72 h. Representative IF staining with anti-EGFR and anti-MTCOI antibodies is shown in the left panel. The expression level of Tid1 in the transfected cells was analyzed by Western blotting, and is shown in the middle panel. β-Actin was used as a loading control. The scores for the colocalization of EGFR and MTCOI in~200 cells was analyzed using the MetaMorph software (right panel). The data shown are the means±SD from three independent experiments; *P<0.05, as assessed with the Student’s t-test.