Fig 2: Effect of Nrf2 on UGT1A expression induced by SFN. The Nrf2 gene was knocked down in SW480 and HT-29 cells. (A and B) The Nrf2 protein levels in HT-29 and SW480 cells were assessed by immunoblotting. (C) The expression levels of Nrf2 were quantified by ImageJ and illustrated by a bar legend. The statistical significance of the results was analyzed by Student's t-test. ****P<0.0001 vs. NC shRNA. (D) The transcriptional level of Nrf2 was quantified by RT-qPCR and illustrated by a bar legend. ****P<0.0001 vs. NC shRNA. The statistical significance of the results was analyzed by Student's t-test. (E and F) The expression levels of UGT1A in HT-29 and SW480 cells decreased in Nrf2-knockdown cells, and the induction effect of SFN on UGT1A expression decreased upon Nrf2-gene knockdown. (G and H) The UGT1A expression in various groups presented in the images of parts E and F was quantified by ImageJ. The statistical significance of the results was analyzed by two-way analysis of variance. *P<0.05, **P<0.01, ***P<0.001 vs. the SFN 0 µM group; ##P<0.01, #####P<0.0001 vs. the NC shRNA group. The results are expressed as the mean ± standard deviation of 3 independent experiments. SFN, sulforaphane; Nrf2, nuclear factor, erythroid 2 like 2; NC, negative control; shRNA, short hairpin RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Fig 3: Ganetespib resistance by UGT1A overexpression. (a) To identify UGT1A isoforms that correlate with ganetespib resistance, we quantified the UGT1A gene clusters by selective RT-PCR analysis, and compared with a primer pair that amplifies all isoforms. This analysis revealed that the cluster composed of isoforms 7, 8, 9, and 10 is expressed at a particularly high level in the ganetespib-resistant cell line HT29. Thus, isoform 10 was chosen for overexpression in Figures 4b and c. (b and c) Overexpression of UGT1A10 confers ganetespib resistance. SW480 and HCT116 cells, both sensitive towards ganetespib, were transfected to overexpress UGT1A10. Cells were subsequently treated with ganetespib, (b) their confluence was determined 72 h later by Celigo, and (c) their cell viability was determined by ATP quantification via luciferase as in Figure 3c. *P<0.05, **P<0.01, ***P<0.005 (t-test)

Fig 4: UGT1A-expressing CRC cells fail to decrease HSP90 client protein levels in response to ganetespib, but not to 17-AAG. (a) Ganetespib-sensitive (SW480, HCT116) and -resistant (SW1463, HT29) cells were each treated with ganetespib, followed by immunoblot analysis of the HSP90 clients AKT and Wee1. Ganetespib addition reduced the levels of HSP90 clients only in sensitive cells, indicating that resistant cells preserve HSP90 function despite ganetespib. (b) Cells were treated and analyzed as in (a), but 17-AAG was used instead of ganetespib. Immunoblot analysis revealed that all cells except the 17-AAG-resistant SW480 cells (see Figure 2c) reduced the levels of HSP90 clients. (c) Knocking down UGT1A protein levels by siRNA in the originally ganetespib-resistant HT29 cells destabilizes HSP90 clients upon treatment with ganetespib in a dose-dependent manner. HT29 cells were first transfected with siRNA for 24 h, followed by incubation with ganetespib for 48 h at the indicated concentrations and immunoblot analysis of HSP90 clients. To avoid complete ganetespib turnover, ganetespib-containing media were renewed every 8 h during the incubation period. (d) Overexpression of UGT1A10 in ganetespib-sensitive cells stabilizes HSP90 clients despite the presence of the drug. HCT116 cells (top) or SW480 cells (bottom) were transfected with the overexpression vector or ß-Galactosidase (ßGal) vector as a control, and subjected to the indicated concentration of ganetespib for 48 h before harvesting. (a–d) GAPDH as a loading control

Fig 5: The metabolic activities, protein expression ratio and mRNA expression ratio of UGT1A9 in adjacent normal and HBV-positive HCC tissues.(A) Metabolism kinetics of propofol in commercial, adjacent normal and HBV-positive HCC HLMs (n = 5). (B) Protein expression ratio of UGT1A9 in adjacent normal (N) and HBV-positive HCC (T) HLMs. Calnexin was applied for protein loading normalization. (C) mRNA expression ratio of UGT1A9 in adjacent normal (N) and HBV-positive HCC (T) tissues. GAPDH was used for normalization. Each data point represents the average of three determinations. Representative data from three independent experiments are shown as the mean ± S.D, *, P<0.05; **, P<0.01; ***, P<0.001. (D) The pairwise correlation of UGT1A9 between metabolic activity reduction ratio, protein expression reduction ratio and mRNA expression reduction ratio. (E) The pairwise correlation of UGT1A9 between metabolic activities, protein expression ratio and mRNA expression ratio in the adjacent normal tissues or HBV-positive HCC tissues. Bivariate correlation analysis was used to analyze the relationship of all indexes (95% confidence interval).