Biorevo fluorescence microscope

Cells of each cell type were seeded into the wells of a Lab-Tek II Chamber Slide System (Thermo Fisher Scientific Inc., Waltham, MA, USA). After transfection with a given MIRNA, including control RNA, the cells were immunostained with an anti-LC3B antibody according to the immunofluorescence protocol of CST. Next, the nuclei were stained with Hoechst 33342, and for actin labeling, the cells were incubated with a fluorescent F-actin probe, rhodamine phalloidin (Cytoskeleton Inc., Denver, CO, USA). Finally, the cells were observed with a BIOREVO fluorescence microscope (Keyence, Osaka, Japan).

Cells were treated with increasing concentrations of MC3 for 24 h and fixed with 4% PFA at room temperature for 15 min and blocked with blocking buffer (5% goat serum, 1% BSA and 0.3% Triton X-100 in PBS) for 30 min. After aspirating the blocking buffer, cells were incubated with cleaved Caspase3 antibody (1:200, Cell Signaling, NEB, Germany) for 1 h. The secondary antibody (Goat anti-rabbit Alexa Flor 594; Dianova, Germany) was added and incubated for 30 min. Hoechst 33342 (1 µg/mL in PBS) was used to visualize nuclei. Images were taken on BIOREVO fluorescence microscope (BZ9000, KEYENCE). Image J software (the particle count function) was used to calculate the percentage of viability, as we described previously¹⁷ .

As described previously [17 (link)], after culturing PBMNCs for 7 days in the endothelial cell growth medium (EGM-2 MV BulletKit, Lonza, Walkersville, MD, USA), we evaluated enrichment of the EPC lineage by staining with Fluorescein Ulex Europaeus Agglutinin I (UEA-I Lectine, FL-1061, Vector Laboratories Inc. Burlingame, CA, USA) and acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (DiI-Ac LDL, BT-902, Biomedical Technologies Inc. St. Stoughton, MA, USA), and then observed the cells under the Bio Revo fluorescence microscope (BZ-9000, Keyence, Osaka, Japan). EGM-2-MV complete medium was adjusted to EBM-2 basal medium by adding 5% FBS (SAFC Biosciences Inc., Lenexa, KS) and supplemented with growth factors, except hydrocortisone. PBMNCs were adjusted to the similar cell density (1 × 10⁶ cells/mL) with EGM-2-MV complete medium containing 5% FBS. Cells were then plated on 6-well Primaria tissue culture plate (2 × 10⁶ cells/2 mL per well) and cultured.

Flow cytometry analysis of JC1 staining was performed as previously described (21 (link)). Briefly, the three CRC cells were incubated with 2 μM of JC1 for 15 min, at the end of the respective treatments. FACS analysis was immediately performed by Guava easyCyte HT sampling flow cytometer. Additionally, MMP was determined using the BIOREVO fluorescence microscope (BZ9000, KEYENCE).

For the histological analysis of atherosclerosis plaques, dissected hearts were fixed in 4% PFA for 4–5 hours and incubated in 30% sucrose at 4 °C overnight. Fixed hearts were horizontally cut, and the half containing the apex was frozen in optimal cutting temperature (O.T.C.) embedding medium (Sakura Finetek, Japan). Serial sectioning was performed in the area of the aortic root by cryotome (Leica, Germany). Each section was 6 µm thick. Hematoxylin and eosin staining and ORO staining were performed as described in established protocols^{3 (link),14 (link)}. For immunohistochemical analysis of Mac2 expression in the plaque macrophages, frozen sections were incubated with a 1:200 dilution of rat anti-mouse Mac2 antibody (Cedarlane, Canada). For secondary fluorescent staining, a 1:500 dilution of Alexa Fluor 488-conjugated chicken anti-rat IgG secondary antibody was used (Invitrogen, USA). Stained histological sections were observed with a BioRevo fluorescence microscope (Keyence, Japan). iRFP fluorescence expression was observed in consecutive, unstained sections under a Cy5.5 filter using a fluorescence microscope (Olympus, Japan).

253J B-V cells were incubated with miR-145 for 48 h and then immunostained with PKM2 antibody according to the immunofluorescence protocol of Cell Signaling Technology. The nuclei were stained with Hoechst 33342, and for actin labeling the cells were incubated with the fluorescent F-actin probe Rhodamine Phalloidin (Cytoskeleton, Denver, CO, USA). The cells were viewed with a BIOREVO fluorescence microscope (Keyence, Osaka, Japan). Standard immunohistochemical staining in clinical samples was performed using anti-KLF4 antibody (Santa Cruz Biotechnology and Proteintech Group, Inc, Rosemont, IL, USA).