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. 2022 May 23;10(5):e004258. doi: 10.1136/jitc-2021-004258

Figure 5.

Figure 5

SIK3 sustains tumor-promoting gene signature after TNF stimulation (A) Two-dimensional hierarchical clustering of 386 genes that were significantly regulated by TNF after 4 hours and significantly affected by SIK3 knockdown. The right panel shows representative gene enrichment analysis for genes having reduced or missing induction by TNF after SIK3 knockdown (fold change ≥2, normalized counts per million >2, false discovery rate (FDR) ≤0.05). (B) qPCR for the detection of NF-κB target genes on 100 ng/mL rHuTNF treatment and SIK3 knockdown in PANC-1 cells. Data were normalized to GAPDH. (C) Assessment of protein expression of NF-κB target genes on 100 ng/mL rHuTNF treatment and SIK3 knockdown in PANC-1 cells. Left panel: representative WB experiment, right panel: relative quantification of protein expression normalized to GAPDH. (D) Luciferase-based cytotoxicity assay for the impact of the overexpression of NF-κB target genes in SIK3 knockout PANC-1 cells. Overespression was performed as described in online supplemental material and methods. Afterwards, cells were transfected with SIK3 siRNA for 72 hours and subsequently subjected to 100 ng/mL rHuTNF for 24 hours. (E) Volcano plot showing differentially expressed genes (from RNA-seq, rHuTNF treatment in siCtrl vs siSIK3 cells) that also have a significant differentially accessible region (determined by ATAC-seq) in their vicinity are highlighted and colored due to their role in tumor biology (see also online supplemental tables S3 and S4).(B) Representative data of at least two independent experiments. (C) Cumulative data of two independent experiments. (D) Cumulative data of at least three different experiments. WB quantifications were obtained by combining two independent experiments. Columns show mean±SEM. P values were calculated using two-tailed Student’ t-test. *P<0.05, **P<0.01. ATAC-seq, assay for transposable-accessible chromatin with sequencing; EV, empty vector; NF-κB, nuclear factor kappa B; ns, not significant; rHuTNF, recombinant tumor necrosis factor; SIK3, salt-inducible kinase 3; TNF, tumor necrosis factor.