Figure 7: CEP peptide disrupts binding of CEP170 and TBK1 and provokes mitotic defects.

(a) H460 or H1650 cell lysates were incubated with 5 μM CEP or scrambled peptide along with His-tagged CEP170 beads. Western blotting shows that binding of TBK1 to CEP170 was specifically disrupted by the CEP peptide, but binding of His-CEP170 to alpha tubulin was not. (b) Scrambled or CEP peptides (1 μM) were preincubated with TBK1 for 15 min and used in a kinase reaction with His6-CEP170 as a substrate. In vitro kinase assay showed that the CEP170 peptide effectively inhibits His-CEP170 phosphorylation by TBK1 but not the phosphorylation of Histone H1. (c) In vitro kinase assays showing that CEP-peptide specifically inhibits TBK1-mediated phosphorylation of CEP170 but not PLK1, where a kinase dead PLK1 was used as substrate. (d) PLK1-mediated phosphorylation of CEP170 is not affected by CEP170-peptide in kinase assays; His6-tagged CEP170 was used as a substrate. (e) CEP170 peptide conjugated to penetratin (CEP-Pen) inhibited the CEP170–TBK1 interaction in H460 cells but a scrambled peptide–penetratin conjugate did not, as seen by a proximity ligation assay. Green signal indicate points of co-localization between TBK1 and CEP170. Scale bar, 10 μm. (f) Quantification of signal from cells treated with peptides (*=P<0.05, t test; Error bars represent s.d. Two replicates were included). (g) H1650 or H460 cells treated with CEP peptide conjugated to penetratin (CEP-Pen) showed reduced the number of mitotic cells versus those treated with scrambled peptide–penetratin conjugate (SCR-Pen) (*P<0.05, t test; Error bars represent s.d. Two replicates were included). (h,i) H460 and A549 cells treated with CEP-penetratin conjugate showed mitotic defects. Phospho-TBK1 (green), CEP170 (red) and DNA (DAPI, Blue). Scale bar, 10 μm. (j) A549 cells treated with CEP-Pen subjected to cold temperature (4 °C) and shifted to 37 °C resulted in more stable microtubules compared to SCR-Pen-treated cells. Alpha tubulin (green), CEP170 (red) and DNA (DAPI, Blue). (k) Microtubules were more stable in A549 cells treated with CEP-Pen when exposed to 0.1 μM nocodazole for 15 min versus SCR-Pen treated cells. Alpha tubulin (green), CEP170 (red) and DNA (DAPI, Blue). Scale bar, 10 μm.