Figure 5. SI113-dependent SGK1 inhibition affects RANBP1 and RANGAP1 transcription, influencing mature and precursor microRNA levels both in vitro and in vivo.

(A) Quantitative RT-PCR analysis of RANBP1 and RANGAP1 mRNA levels in SI113-treated HUH7 cells compared with control cells. The results, normalized to the HPRT housekeeping gene, are represented as the fold expression ± SD of triplicate assessments and were evaluated using the t-test. (B) Western blot analysis of proteins from HUH7 cells with or without 12.5 μM SI113 treatment for 72 h. Cell extracts (50 μg aliquots) were assessed via SDS-polyacrylamide gel electrophoresis, followed by immunoblotting using a RANBP1 antibody, a RANGAP1 antibody and a GAPDH antibody. (C) Quantitative RT-PCR analysis of RANBP1 and RANGAP1 mRNA levels in tumor tissues of HCC xenografts in NOD/SCID mice with or without SI113 treatment. The HCC mouse xenograft model and treatment regimen were developed as previously reported⁵⁴. (D) Western blotting analysis of proteins from mouse tumor tissues with or without SI113 treatment. (E) Quantitative RT-PCR analysis of both mature and precursor microRNAs in mouse tumor tissues with or without SI113 treatment. The values are plotted as Log₁₀ of fold expression ± SD and evaluated using the t-test. The assay was performed as described above. Statistical significance is reported at the top of the graph. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Cropped gels are shown and full-length gels are included in SFs. Results are the average ± SD of three independent experiments each run in triplicates.