Fig. 6. Increased HAS2 leads to doxorubicin resistance by upregulating NRF2 signaling in gastric cancer SNU620-DR cells. (A) Cell viability was monitored after doxorubicin incubation for 72 h in SNU620 and SNU620-DR cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Values represent the mean ± SD from 8 sampled wells. ap<0.05 compared with SNU620. (B) NRF2, AKR1C1, and CD44 protein levels were monitored in SNU620 and SNU620-DR (DR) cells using western blotting analysis. (C) CD44 expression levels were determined in SNU620 and DR cells using flow cytometry analysis. (D) HAS2 transcript levels were determined in SNU620 and SNU620-DR cells by relative quantification real-time PCR analysis. HPRT1 was used as a housekeeping control gene. Data represent ratios with respect to SNU620 and are reported as the mean ± SD of two experiments. (E) HA concentration was measured in SNU620 and SNU620-DR cells using ELISA. Values represent the mean ± SD of duplicated sample measurements. ap<0.05 compared with SNU620. (F) SNU620-DR was transfected with non-specific control RNA (siCTRL) or HAS2-specific siRNA (siHAS2), and NRF2 protein levels were monitored. (G) SNU620-DR was incubated with vehicle (veh) or 4-MU (5 mM) for 24 h, and NRF2 protein levels were monitored. (H) Cell viability was monitored after doxorubicin and 4-MU incubation in DR cells using MTS assay. Values represent the mean ± SD of duplicate sample measurements. ap<0.05 compared with vehicle (veh) group. In the case of western blot results, similar blots were obtained in at least three experiments.
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