Figure 3. Alteration of gene expression profile in cSCC cells afterAIM2 knockdown.

(A) cSCC cells (UT-SCC7) were transfected with AIM2 siRNA_5, AIM2 siRNA_6, or control siRNA (CsiRNA) (75 nM) and transfection efficiency was examined by Western blotting 72 hours after transfection. β–Actin was used as a marker for equal loading. (B-E) Three cSCC cell lines (UT-SCC7, UT-SCC12A, and UT-SCC105) were transfected with AIM2 siRNA_6 or control siRNA. 72 hours after transfection, whole transcriptome analysis was performed with RNA-seq. Summary of (B) Ingenuity pathway analysis (IPA) biofunctions and (C) gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to AIM2 knockdown (P < 0.05, FC log2 > 0.75). (D) Molecular interactions involved in the network Cell cycle, cellular assembly and organization, DNA replication, recombination, and repair as one of the top networks regulated after AIM2 knockdown in cSCC cells (score=24; Supplementary Table 1) are depicted with IPA (P<0.05 and fold change (FC) (log2) >0.75). Red color indicates up-regulation, and green color indicates down-regulation. The intensity of the color shows the magnitude of FC. The arrows and the lines show the interactions (solid line indicated direct and dashed line indirect interaction). (E) Normalized CDK1 mRNA expression was determined from cSCC cells with RNA-seq after AIM2 siRNA_6 knockdown (left panel). UT-SCC7 cells were transfected with AIM2 siRNA_5, AIM2 siRNA_6, or control siRNA (75 nM) and expression of Cdk1/Cdk2 was determined by Western blotting 72 hours after transfection with siRNAs. Level of Cdk1/Cdk2 was quantitated by densitometry and corrected for the level of β–Actin used as a marker for equal loading (right panel).