μ dishes

To provide proof-of concept that the pharmacological inhibitor CCG-1423 prevents MRTF-A translocation to the nucleus during all stages of C2C12 cells differentiation, 2.0 × 10⁴ or 2.0 × 10⁵ myoblasts were plated in 6 well plates or 35 mm μ-Dishes (ibidi, Munich, Germany), respectively. Myoblasts were allowed to growth for 48 h in growth medium as described above, following what the medium was replaced by serum-free DMEM supplemented with either 5 μM CCG-1423 (Merck Millipore, Kilsyth, VIC) or dimethyl sulfoxide (DMSO) at a final concentration of 0.1%. Similarly, myotubes were differentiated as described above and were serum-starved after either 48 or 96 h of differentiation. Following overnight incubation, the medium was changed to DMEM supplemented with 20% FBS containing the same concentrations of CCG-1423 or DMSO, respectively. After 1 h, the cells were prepared for immuno-localisation.

To visualize cellular morphological changes, all cells cultured in their corresponding pHe medium were collected and plated in 35-mm diameter μ-dishes (Ibidi, Martinsried, Germany). A minimum of 10 randomly chosen microscopic fields (~ 70 cells per field) were imaged using an AF6000 LX microscope (Leica Microsystems, Singapore) equipped with Zyla sCMOS camera (Andor by Oxford Instruments, Belfast, UK) and objective lens (HCX PL Fluotar L 20x/0.40 CORR PH1) with 1.8x digital zoom. To observe mitochondrial morphology and cristae ultrastructure, the TEM (transmission electron microscopy) images of each cell group were taken on an H-7650 electron microscope (Hitachi High-Technologies, Tokyo, Japan) equipped with a CCD (charge-coupled device) camera (Advanced Microscopy Techniques, Danvers, MA, USA). The density of cristae was calculated based on 45 randomly selected mitochondria per cell group with ImageJ software (https://imagej.nih.gov/ij).

HUVECs (80,000 cells/well) or HRMECs (40,000 cells/well) were seeded and cultured on the culture-inserts of μ-dishes (Cat# 81176, Ibidi) until reaching confluence. The culture-inserts were subsequently removed to generate wound gaps. Fresh EBM-2 medium (supplemented with 0.1% FBS) was added with 2.5 nM VEGF^{29 (link),30 (link)} or the indicated concentrations of ginsenosides. After 12 hr and 24 hr, the migrated cells within the wound were monitored with a cell analyzer, JuLITM (Cat#JULI-B004, NanoEnTek). Cell migration was quantified by measuring the ratio of the migration area to the total area of the wound gap using ImageJ software (NIH).

HO1N1 cells were grown in 10 mm μ-Dishes (Ibidi GmbH) at a density of about 1 × 10⁴ cells per well. Prior to imaging, nuclear DNA was stained with NucBlue™ live cell stain according to the manufacturer’s protocol (Life Technologies, Grand Island, NY, USA). The cell membrane was labeled using PKH67 Green Fluorescent Cell Linker Kit (Sigma–Aldrich), according to the manufacturer’s instructions. Early endosome (EE) and late endosome (LE) were labeled using Invitrogen™/CellLight™ Early Endosomes-GFP, BacMam 2.0 (C10586, Invitrogen) and Invitrogen™/CellLight™ Late Endosomes-GFP, BacMam 2.0 (Invitrogen C10588, Invitrogen), following the manufacturer’s protocols. The staining solution was replaced with culture media and cells were immediately imaged. Confocal stacks were achieved with CLSM system (TCS SP8, Leica) using a 63 × 1.4 NA oil objective, and processing was completed using a 5% CO₂ incubator at 37 °C. Time-lapse images of the living cells were recorded every 5 s. Fiji software was used for quantification.^{64 (link)}

The Xfect Protein Transfection kit (Takara Bio Inc., Shiga, Japan) was used to introduce the peptides into cells according to the manufacturer’s instructions. Briefly, cells were grown to 60–70 % confluence in coverslip-like-bottomed 35-mm diameter culture dishes (μ-Dishes; ibidi, Verona, WI, USA) and incubated at 4 °C for 1 h in serum-free medium supplemented with 0.25 × Xfect protein transfection reagent containing 0.4 μg (for RLE-6TN cells) or 0.2 μg (for the other cells) of either the C-ICD or C-ICDmut peptide labeled with FITC or unlabeled, as indicated. The cells were washed with serum-free medium and incubated in growth medium. The same treatment was applied 24 h later. The cells were subjected to Western blot analyses after an additional 24 h, and mitochondrial labeling or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were conducted.

For isolation of mouse macrophages, mice received an intraperitoneal injection of thioglycollate (4%). Cells were collected after 4 days by peritoneal lavage, centrifuged, resuspended in RPMI medium (10% FBS) and plated onto μ-dishes (Ibidi) for 24 h.
Human THP-1 monocytes were stimulated with PMA (300 nM, Merck) overnight to differentiate them into macrophages, which were then transfected with SR-BI-specific siRNA oligonucleotides or control oligonucleotides (Qiagen) for 48–72 h. Human neutrophils were isolated from whole blood using a density gradient separation method with dextran (3%, MW 500,000, Roth) containing HBSS/Hepes (10 mM) medium. After centrifugation, separation of the neutrophil layer and lysis of residual erythrocytes, the neutrophils were washed and resuspended in RPMI/Hepes (10 mM). Neutrophil apoptosis was induced by overnight incubation with pyocyanin (25 μM, Sigma). The apoptotic neutrophils were stained with TAMRA (50 μM, Thermo Fisher Scientific) and incubated for 30 min with macrophages at 37 °C.
CHO cells were stably transfected with the cDNA for human SR-BI14 (link). Transient transfections with different plasmids for murine SR-BI (native mSR-BI and its mutants SRCDSR and CDSRCD27 (link), donated by Margery Connelly), were performed using Lipofectamine^®2000 (Thermo Fisher Scientific).

The cardiac myocyte‐removed fraction was dispersed in semi‐solid culture medium and cultured in high‐quality plastic dishes (μ‐Dishes; ibidi GmbH, Germany) (Omatsu‐Kanbe and Matsuura 2009). Beating ACMs were identified in the culture after ~5 days.