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. 2018 Jan 1;8(3):676–692. doi: 10.7150/thno.21463

Figure 5.

Figure 5

AKR1C1 interacts with STAT3 to promote metastasis. (A) AKR1C1 increases p-STAT3 nuclear translocation. NCI-H1299 cells stably expressing AKR1C1 were treated with IL-6 for 30 min after starving overnight. Cytoplasmic and nuclear fractions from NCI-H1299 cells were separated and subjected to western blot as indicated. The nuclear marker Lamin B and the cytoplasmic marker GAPDH were used to demonstrate the purity of the fractions. (B) AKR1C1 knockdown decreased endogenous p-STAT3 nuclear translocation. NCI-H460 cells were starved overnight with AKR1C1 interference. Cytoplasmic and nuclear proteins from NCI-H460 cells were collected and subjected to western blot as indicated. (C) NCI-H460-scramble and NCI-H460-shAKR1C1 cells were treated with IL-6 (20 ng/mL) for 30 min after overnight starvation, and then the expression and localization of p-STAT3 and AKR1C1 was detected using immunofluorescence staining with antibodies against p-STAT3 and AKR1C1. (D-E) Twenty-four hours post-transfection, ChIP assay was carried out using anti-HA antibody, and irrelevant anti-IgG antibody was introduced as negative control. The protein-chromatin immunoprecipitates were subjected to PCR (D) and qPCR (E) analyses. (D) Input lane, total genomic DNA used as control for the PCR reaction; IgG lane, negative control. (E) Upper: map of the primers used for amplifying STAT3-binding regions of MMP2, TWIST and SOX2; tss: transcription starting site. Bottom: ChIP-qPCR analysis of 293FT cells transfected with STAT3 or/and AKR1C1, using anti-HA antibody and PCR primers. IgG was used as a negative control. Enrichment of HA-STAT3 on the promoter regions of the target genes was calculated. Data are mean ± SD. (F) Immunoblotting analysis of lysates after immunoprecipitation from NCI-H1299 cells transfected with Flag-AKR1C1 and HA-STAT3. (G) Cucurbitacin I potently suppress the migration promoted by AKR1C1. (H) STAT3 inhibitor, Cucurbitacin I imposed little impact on the viability of NCI-H1299 (low AKR1C1 expression) and NCI-H460 (high AKR1C1 expression). (I) qRT-PCR analysis was used to assess the function of STAT3 upon exposure to Cucurbitacin I. (J) STAT3 knockdown suppressed the motility of NCI-H460 (high AKR1C1 expression). Statistical significance was determined by Student's t-test. *, P < 0.05; **, P < 0.01, ***, P < 0.001.