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. 2016 Mar 17;5:e11288. doi: 10.7554/eLife.11288

Figure 5. Knowledge-based analysis of the microarray data reveals that NIFK regulate CK1α and Wnt signaling.

(A) The relative knockdown efficiencies of Ki-67 in (A) PC13 and (C) H1299 cells. Cell migration and invasion were evaluated via the transwell assay using (B) PC13 and (D) H1299 cells. (E) List of the top 10 signaling pathways altered by NIFK overexpression. The microarray data of NIFK overexpression in PC13 cells were analyzed using the MetaCore Maps database. (F) List of the top 10 networks affected by NIFK overexpression. The gene signatures were processed via the MetaCore Networks database. (G) Representative signature of genes displaying a≥1.5-fold change in expression due to NIFK overexpression in PC13 cells. The red and green bars represent upregulation and downregulation, respectively. (H) Knowledge-based IPA analysis of the microarray data focusing on CK1α (CSNK1A1)-mediated signaling. The red and green circles represent upregulation and downregulation, respectively. (I) The nuclear levels of β-catenin in cells with NIFK overexpression were showed. Nuclear fractions were extracted from A549 and PC13 cells. (J) TCF/LEF transcriptional activity alteration in the indicated H661 and H1299 cells. TOPflash: reporter plasmid with TCF binding sites. FOPflash: reporter plasmid with mutated TCF binding sites.

DOI: http://dx.doi.org/10.7554/eLife.11288.010

Figure 5.

Figure 5—figure supplement 1. NIFK down-regulates CK1α, and up-regulates β-catenin level especially in nucleus.

Figure 5—figure supplement 1.

RT-PCR was performed on RNA samples from PC13 cells overexpressing NIFK.
DOI: http://dx.doi.org/10.7554/eLife.11288.011
Figure 5—figure supplement 2. Phospho-β-catenin levels are decreased after NIFK overexpression.

Figure 5—figure supplement 2.

β-catenin phosphorylation was studied in A549 and PC13 cells. Cells were pre-treated with 20 μM proteasome inhibitor MG132 for 3 hr prior lysate harvest for Western blot.
DOI: http://dx.doi.org/10.7554/eLife.11288.012
Figure 5—figure supplement 3. NIFK regulates downstream transcriptional targets of TCF4/LEF via CK1α.

Figure 5—figure supplement 3.

RT-PCR was performed on the indicated samples. The expression levels of transcriptional targets of TCF4/LEF are presented.
DOI: http://dx.doi.org/10.7554/eLife.11288.013
Figure 5—figure supplement 4. NIFK regulates downstream transcriptional targets of β-catenin.

Figure 5—figure supplement 4.

Microarray analysis of PC13 cells stably overexpressing NIFK by lentiviral infection was performed. β-catenin transcriptional targets upregulated by NIFK were examined and identified via IPA analysis.
DOI: http://dx.doi.org/10.7554/eLife.11288.014