Bf 3001c

EMCMs were grown on collagen-I–coated Bioflex plates (BF-3001C, Flexcell International). EMCMs were concurrently exposed to cyclic stretch of 16% at 1 Hz for 24 hours using a Flexcell FX-5000 Tension system (Flexcell International) and to static condition (control) on Bioflex plates (22 (link)).

As described previously^{32 (link)}, the primary cortical neurons were obtained fromWistar rat fetuses at ED18 and maintained for 8 to 10 days before the experiments. Cerebral cortices were isolated and dissociated by Papain (40 mg/ml) (#5125, Sigma- Aldrich, MO, USA) digestion and trituration with a 10 ml plastic pipette. Primary cortical neurons were suspended in a Neurobasal medium (#21103049, GIBCO/Life Technologies, NY, USA) supplemented with 2% B27 (#17504044, GIBCO/Life Technologies), 100 U/ml penicillin, 100 μg/ml streptomycin (# 15140-122, GIBCO/Life Technologies) and 200 mM GlutaMAX™ Supplement (# 35050-061, GIBCO/Life Technologies). Neurons were plated at a density of 3 × 10⁶/well in 6-well plates (#BF-3001C, FLEXCELL, NC, USA) pre-coated with Poly-L-Lysine (#3438-100-01, R&D Systems, Minneapolis, MN, USA) (0.1 mg/ml). Neurons were cultured up to 7 days in vitro (7 DIV) at 37 °C in a humidified 5% CO₂ incubator. Half the medium was changed every 4 days. The cells were plated at a high density (3 × 10⁶ cells/well) on a poly-l-lysine-coated well and cultured in a Neurobasal medium (Invitrogen).

In order to investigate CASA complex component expression in mechanically loaded PDL cells, static tensile strain was applied to PDL cells cultured to 80% confluence on Bioflex® collagen type I‑coated culture plates with silicone membrane flexible bottom wells (BF-3001C; Flexcell International, Hillsborough, NC, USA). The plates were placed into a strain device (FX-6000T™ Tension System, Flexcell International) provided with a BioFlex baseplate with cylindrical post as loading platform in the dimensions of the flexible-bottom wells. The system comprises a computer-regulated bioreactor using vacuum pressure and positive air pressure to apply a defined, controlled, static deformation to cells growing in monolayer. After cells were seeded into the plates and grown for 24 h before experimentation, continuous cell stretching was performed at 2.5, 5, and 10% [6 (link)]. The BioFlex baseplate with the loading stations and the loading posts was placed in an incubator to provide a humidified 5% CO₂ atmosphere at 37 °C and cells were subjected to mechanical loading for 24 h. Then, plates were removed from the construction and the flexible membranes with the stretched cells were subjected to further experiments as described below. In order to elucidate mechanisms that are provoked by tension-induced CASA, unstretched cells served as controls in each experiment.

Cyclical stretch-relaxation was applied for cultured PASMCs and TASMCs. Cells were grown to 80% confluence and cultured in DMEM with 0.5% fetal bovine serum on six-well silicone elastomer-bottomed culture plates (Bioflex; Flexcell, BF-3001C, Hillsborough, NC, USA) that had been coated with collagen type I. After incubating for 48 h in the quiescent medium, cells were subjected to cyclical stretch/relaxation using the Flexercell Strain Unit FX-4000 (Flexcell). This unit (Figure 3a) is a modified version of the one initially described by Wipff et al [49 (link)]. Under computer control, a vacuum (<15 to 20 kPa) was repetitively applied (60 cycle/min, the maximal stretch of 10% at the periphery) to the silicone elastomer-bottomed culture plates, which were maintained in a humidified incubator with 5% CO₂-95% air atmosphere at 37 °C. Cells were subjected to stretch/relaxation for a variable duration up to 24 h as indicated.

The regimen for the cyclic strain was adapted from a previous publication [58 (link)]. Briefly, HaCaT and SqCC/Y1 cells, with/without Dsg3 knockdown, were plated and grown to confluence for 1~2 days on collagen-coated BioFlex 6-well culture plates with flexible silicone elastomer bottoms (BF-3001C, Flexcell^® International Corporation, Burlington, NC, USA). Each plate was placed over the loading station containing six planar faced posts. Cell monolayers were subjected to equiaxial cyclic mechanical stretching with cyclic strain range of amplitude in 10–15% and a frequency of 5 Hz, in a Flexcell FX-5000 Tension System (Flexcell International for varying durations, i.e., 4–6 h or 24 h. Control cells were seeded in the same BioFlex plates along with the strained cells but maintained at a static state (stationary) without any exposure to mechanical stretch. Cells in the plates after strain were fixed with 3.6% formaldehyde for 10 min and permeabilized in Triton X-100 (0.1% in PBS), or without Triton permeabilization, which preferentially detects peripheral proteins, including those at the junctions [23 (link)], before immunofluorescence and fluorescent microscopy. Alternatively, lysates were extracted either immediately after a strain or transferred to static state and harvested later at different time points, for mRNA analysis by RT-qPCR or protein analyses by Western blotting.