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. 2016 Dec 15;18(1):61–71. doi: 10.15252/embr.201642683

Figure 4. Subcellular localization and protein level of NLK are regulated by cell density.

Figure 4

  1. Localization of endogenous YAP and NLK in low‐density (LD) or high‐density (HD) MCF10A (upper panel) or NIH3T3 cells (bottom panel) was examined by indirect immunofluorescence. Nuclei were stained with DAPI. Scale bars: 20 μm.
  2. Level of NLK decreases in a cell density‐dependent manner. Lysates from low‐density (LD) and high‐density (HD) MCF10A, HEK293T, and NIH3T3 cells were immunoblotted with antibodies indicated in the figure.
  3. Level of endogenous NLK mRNA is unaffected, whereas the expression of YAP target genes was reduced, at high cell density. Quantitative real‐time PCR analyses for expression of AMOTL2, CTGF, CYR61, and NLK in low‐density (LD) and high‐density (HD) NIH3T3 cells were performed. Quantification of AMOTL2, CTGF, CYR61, and NLK mRNA was normalized with the level of Gapdh. Data represent average values from a representative of multiple experiments performed in triplicate. Error bars indicate standard deviations of triplicate measurements. Data are presented as mean ± SD. *P < 0.05 and ***P < 0.005. Student's t‐test was used for statistical analysis.