Fluorescence microscopy

Myocardial tissue from INS^C94Y transgenic diabetic pigs and non-diabetic littermates were obtained from Institute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich [2 (link)]. Cryosections were stained for ADAM10 (Abcam) and PECAM-1 (Thermo Fischer, Waltham, MA, USA), 1:100 dilution, followed by Alexa-488 and Alexa-594 secondary antibody (Thermo Fischer). Forty times magnification images were taken by fluorescence microscopy (Leica Microsystems, Germany) and analyzed by its software system.
Endothelial tube networks were fixed with 4% paraformaldehyde, washed with PBS and blocked for one hour with 5% bovine serum albumin (BSA). A total of 20 μg/mL rabbit anti-ADAM10 antibody (Invitrogen) was prepared in 5% BSA and incubated with the cells for 8 h, washed with PBS, incubated with Alexa Fluor-488-coupled secondary goat anti-rabbit antibody (Invitrogen) for 2 h and finally washed with PBS. The 10× or 20× images were taken by fluorescence microscopy (Leica Microsystems, Germany).

The CCK-8 assay was performed as we described earlier [27 (link)]. When running the assay, reagents from a CCK-8 reagent-based kit (Promega, USA) absorbance at 450 nm was measured using a plate reader (BioTek, USA). Cell proliferation was detected using the incorporation of 5-ethynyl-29-deoxyuridine (EdU) with the EdU Cell Proliferation Assay Kit (Invitrogen, USA) according to the manufacturer’s protocol. The EdU proliferation assay was performed as described earlier [26 (link)]. The proportion of cells that incorporated EdU was determined using by fluorescence microscopy (Leica, German).

Huh-7 and Sk-hep-1 cells were treated with 0, 5, 10 and 20 μM of dehydrocrenatidine, followed by fixing with 4% paraformaldehyde and staining with DAPI reagent (1:10,000, in triton-X100 mixed with 1X PBS) in the dark for 30 min. Finally, the cells were photographed using fluorescence microscopy (Lecia, Bensheim, Germany). The chromosome condensation and fragmentation were observed and quantified.

The cells were fixed with 4% paraformaldehyde for 30 min and then kept stable in 0.2% Triton X-100 for 30 min to rupture cell membranes. Subsequently, the cells were blocked with 2% bovine serum albumin for 30 min. The cells were then incubated overnight at 4 °C with antibodies against BAP31 antibody (1:200, Abcam, Cambridge, UK) and β-catenin (1:200, Proteintech, Wuhan, China). After washing, the cells were further incubated with the appropriate fluorescence-conjugated secondary antibody (CST, Danvers, MA, USA) for 1 h at room temperature and stained with DAPI (Beyotime). They were then examined by fluorescence microscopy (Leica).

LPC006 and LPC067 cells were transfected with 100 nM of anti-miR-NC, anti-miR-100, or anti-miR-125b and the next day were treated with 1 µM gemcitabine (GEM) chemotherapy for 24 h. Following treatments, cells were washed twice with PBS and fixed in 4% PFA for 15 min. Cells were then resuspended in a solution containing 8 μg ml⁻¹ bisbenzimide HCl and incubated for 15 min. Cells were spotted on glass slides and were examined by fluorescence microscopy (Leica, Wetzlar, Germany). A total of 200 cells from randomly chosen microscopic fields were counted, and the percentage of cells displaying chromatin condensation and nuclear fragmentation relative to the total number of counted cells (apoptotic index) was calculated. The average percentage of apoptosis induced by GEM and anti-miR-NC (negative control) was 19% and 12% in the LPC006 and LPC067 cells, respectively. These values were set as 100%, in order to show the difference after treating with the other anti-miRs more clearly. Apoptosis induction <20% indicates that both cell lines are not very sensitive to GEM.