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. 2016 Sep 10;5(3):35. doi: 10.3390/cells5030035

Figure 6.

Figure 6

LPS, as a model for in vitro activation of inflammatory signaling pathways, upregulates K8 and K19 and increases phosphorylation of K8 at S74. Colorectal cancer HT-29 cells were treated with 500 ng/mL and 1000 ng/mL LPS for 48 h to induce inflammatory stress signaling. (A,B) LPS induced stress-activated inflammatory signaling, as seen by decreased levels of the NF-κB inhibitor IκBα, which was quantified by Western blotting and normalized to Hsc70. HT-29 cells showed an upregulation of the major colonic keratins K8 and K19 following treatment with 1000 ng/mL LPS. An increase in K8 pS74 was seen after treatment with 500 ng/mL LPS. ** p < 0.01, *** p < 0.001. (C) The upregulation of K8 (panels a, d and g; green) and the increase in K8 pS74 (panels b, e and h, and further magnified from white boxes to c, f and i, red) in response to LPS-treatment were confirmed by immunofluorescence staining. Nuclei (DNA) are shown in blue. Scale bar = 25 μm for a, c, d, f, g and i. Scale bar = 75 μm for b, e and h. (D) The increase in K8 pS74-positive cells was quantified, and showed a 6.3% increase in the amount of K8 pS74-positive cells after 500 ng/mL LPS-treatment compared to non-treated cells. For 1000 ng/mL LPS-treatment, the corresponding increase compared to non-treated cells was 3.1%. Numerical data are shown as average ± SD. Statistical significance for Western blot and immunofluorescence data was determined by t-test.