Thermo block

In situ hybridization was performed as described previously [28 ]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), paraffin cross-sections (5-μm thick) from clinical PC tissues were deparaffinized and rehydrated for fluorescence in situ hybridization. Hybridization was performed using fluorescence-labeled miR-193a-5p probes with hybridization buffer (Exiqon) by incubation at 56 °C for 1 h in a thermo-block (Labnet, USA). After stringent washing with SSC buffer, nonspecific binding sites were blocked with 10% normal goat serum (710,027, KPL, USA). According to need, the sections were then incubated for 1 h at 37 °C with anti-HO-1 primary antibody (ab13248, Abcam) or anti-Bach2 (ab83364, Abcam) diluted 1:50 in PBS or incubated with secondary antibody directly. After washing with PBS, the sections were incubated with a rhodamine-labeled secondary antibody (031506, KPL, USA) at 37 °C for 30 min. Images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica).

Stock solutions of the studied compounds were made at a concentration of 1 mM in DMSO:MeOH mixture (10∶90 v/v) to prevent their precipitation. For biotransformation incubations, the solution of compounds was diluted to 10 µM with the presence of 1 mM of NADPH (Sigma-Aldrich, St. Louis, MO, USA) in a potassium phosphate buffer (0.1 M, pH 7.4). Incubation was carried out in thermoblock (Labnet International, Edison, NJ, USA) at 37°C and started with the addition of pooled human liver microsomes with a final concentration of 0.53 mg/mL (Sigma-Aldrich, St. Louis, MO, USA). Directly after HLM was added and at 120 min of incubation the reaction was ended by the addition of an equal volume of ice-cold methanol with 0.1% (v/v) of formic acid. The samples were immediately centrifuged (10 min at 11700 g) and the resulting supernatant was directly analyzed or kept at −80°C until LC-MS or LC-QTOF-MS analysis.
Additionally, chemical stability (negative control) in biotransformation experiment conditions was performed (phosphate buffer, 37°C, 2 h) to evaluate possible instability, unrelated to the activity of microsomes (including putative isomerization).

In situ hybridization was performed as described previously [18 (link)]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), cultured cells smears were deparaffinized and rehydrated for fluorescence in situ hybridization (FISH). Hybridization was performed using fluorescence-labeled miR-140-5p probes with hybridization buffer (Exiqon) by incubation at 56°C for 1 h in a thermo-block (Labnet, U.S.A.). After stringent washing with SSC buffer and PBS, images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica). miR-140-5p probe was purchased from GenePharma Co., Ltd (Shanghai, China). The sequence of miR-140-5p-cy3-probe was as follows: TACCATAGGGTAAAACCACTG. The in situ hybridization image analysis was done according to the protocol of FISH image analysis in clinical diagnosis. Briefly, 200 cell nuclei were (stained with DAPI) counted under fluorescence microscopy. The FISH-positive cells were stained with red probes and emitted red fluorescence. Positive-cell numbers (per 200) cells were used to statistical analysis. Each experiment was repeated three times.