Ab192785

To verify the results of this study, we collected 54 paired tumor tissues and adjacent tissues from our hospital to carry out immunohistochemistry. Immunohistochemistry was performed as described previously (24 (link), 25 (link)) using anti-AKR1C1(ab192785, Abcam) and anti-CARS1(ab126714, Abcam) antibodies. In brief, colon cancer tissues and adjacent tissues sections were deparaffinized in xylene and rehydrated with ethanol. Heat mediated antigen retrieval was performed in 10 mM citrate buffer at pH 6.0. After blocking with normal goat serum, slides were incubated with primary antibodies against AKR1C1 and CARS1 overnight (4°C). Tissue sections were then stained with biotinylated secondary antibody (Vector lab) for 1 h at room temperature, followed by the Vectastain Elite ABC reagent (Vector lab) for 30 min. The peroxidase reaction was developed with diaminobenzidine (DAB kit; Vector lab) and the slides were counterstained with hematoxylin (Sigma).
The positive expression of AKR1C1 and CARS1 were mainly located in the cytoplasm. Positive cells accounted for a percentage score standard: 0 = no positive cells; 1 = 1–25% positive cells; 2 = 26–50% positive cells; 3 = 51–75% positive cells; 4 = more than 75% positive cells.

Western blot 24 h after stress treatment human KCs were washed twice with PBS and then harvested with lysis buffer (70 mM Tris-HCl, pH6.8, 1,1%SDS, 11,1% (v/v) glycerol, 0005% bromophenol blue (BioRad)) containing protease inhibitor cocktail (Abcam, Cambrige, UK) and Pierce TM Phophatase Inhibitor Mini Tablets (Thermo Fisher Scientific, Waltham, MA) on ice and immediately sonicated. Immunoblotting using antibodies for CDK1 (1:1000; ab32384, Abcam), AKR1C1 (1:1000; ab192785, Abcam), HMOX1 (1:1000, ADI-SPA-896, Enzo), and GAPDH (1:2000; clone 5G4; HyTest Ltd., Turku, Finland), was performed as previously described [34] (link). As secondary antibody, goat anti-rabbit IgG-HRP (Biorad 170–6515) or sheep anti-mouse IgG-HRP (NA-931-V, GE Healthcare, Little Chalfont, UK) were used and subsequent chemiluminescent quantification on ChemiDoc imager (Bio-Rad) was performed. The signal was measured with Image Lab 4.1 analysis software (Bio-Rad) and target bands were normalized to GAPDH.