Fig 2: SIK2 inhibitors enhance olaparib sensitivity in ovarian cancer and breast cancer cells.(A and B) Dose-response curves for ARN3236 or ARN3261 (blue), olaparib (green), or ARN3236 or ARN3261 combined with olaparib (red) for 96 hours in 12 cancer cell lines (A) and 3 nonmalignant cell lines (B). The IC50 of inhibitors and concentration ratio of SIK2 inhibitors to olaparib used in each cell line are listed in Supplemental Table 2. The statistical significance between olaparib alone and SIK2 inhibitor combined with olaparib was calculated with 2-way ANOVA and Tukey’s multiple-comparison test. NS, P > 0.05; ***P < 0.001; ****P < 0.0001 (red stars indicate SIK2 inhibitor + olaparib enhancing the effect of olaparib alone; blue stars indicate SIK2 inhibitor + olaparib inhibiting olaparib’s effect). A combination index (CI) at ED90 (determination of the 90% effective dose) was calculated using CalcuSyn software. Experiments were repeated 3 times. Representative data were from 1 independent experiment with 4 technical repeats.

Fig 3: Expression level of SIK2 in CRC lines. (A) mRNA expression levels of SIK2 were notably increased in all types of cancer in the Cancer Cell Line Encyclopedia. The expression levels of SIK2 in the normal colonic epithelial cell line, NCM460, and different CRC cell lines (LoVo, SW480, SW620, CW2 and HCT116) were determined via (B) western blotting and (C) reverse transcription-quantitative PCR. ***P<0.001 vs. NCM460. CRC, colorectal cancer; SIK2, salt-inducible kinase 2.

Fig 4: SIK2 silencing attenuates glycolysis in colorectal cancer cell lines. The levels of (A) pyruvate kinase activity, lactate production and (B) glycolysis were detected using their corresponding kits. The mRNA and protein expression levels of the glycolysis-related genes, Glut1, HK2 and PKM2, were determined via (C) reverse transcription-quantitative PCR and (D) western blot analyses, respectively. **P<0.01, ***P<0.001 vs. siRNA-NC group. PKM2, pyruvate kinase M2; Glut1, glucose transporter 1; HK2, hexokinase 2; siRNA, small interfering RNA; NC, negative control.

Fig 5: Association between SIK2 and TRIM28. The mRNA and protein expression levels of TRIM28 in NCM460, LoVo, SW480, SW620, CW2 and HCT116 cells were measured using (A) reverse transcription-quantitative PCR and (B) western blot analyses, respectively. ***P<0.001 vs. NCM460. (C and D) Co-immunoprecipitation assays were conducted to verify the association between SIK2 and TRIM28. TRIM28 (E) mRNA and (F) protein expression levels in HCT116 cells. ***P<0.001 vs. siRNA-NC group. SIK2, RNA salt-inducible kinase 2; TRIM28, tripartite motif containing 28; siRNA, small interfering RNA; NC, negative control.