P30 3302

Drosophila S2 cells (R69007, Thermo Fisher Scientific, Schwerte, Germany) were grown at 28°C in complete Schneider’s Drosophila Medium (P04-90500, PAN-Biotech, Aidenbach, Germany), supplemented with 10% fetal bovine serum (P30-3302, PAN-Biotech, Aidenbach, Germany), 50 U/ml Penicillin/Streptomycin, 14.6 mg/ml Glutamine (10378016 Thermo Fisher Scientific, Schwerte, Germany). For routine passages, cultures were split 1:5 when they reached 90% confluence, generally every 3–4 days.
For cell aggregation assay, 2 ml S2 cells (1−3 × 10⁶) at the fifth to 10th passage were seeded into six-well plates on the day before transfection. Cells were transiently transfected using Effectene Transfection Reagent (301425, Qiagen, Hilden, Germany) and incubated at 28°C for 3 days. Average transfection efficiency was 9.5% for 500 ng of the GFP-Beat-Cherry construct. Transfected cells were seeded in a total volume of 600 μl into a round glass bottom dish (35 mm diameter; ibidi GmbH, Martinsried, Germany) and incubated at room temperature for 2 h with constant shaking at 100 rpm on a rocking platform (VWR International, Leuven, Belgium). Glass bottom dishes were examined for cell aggregates using confocal microscopy.

HEK293T, A549, DF1, and MDCK (Madin-Darby canine kidney) cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and MDCK MAVS knockout cells, maintained in RPMI 1640 (SH30809.01, HyClone) medium or Dulbecco’s modified Eagle’s medium (Gibco, NY, USA) or Ham’s/F-12 (SH30026.01, HyClone) medium supplemented with 10% heat-inactivated fetal bovine serum (522 PAN-Biotech, P30-3302), and incubated in 37°C humidified incubator with 5% CO₂.

For experiments, we seeded 70,000 PDLF up to the sixth passage either on polystyrol plates (353046, BD Bioscience) for pressure application or on collagen-coated bioflex plates (BF-3001C, Dunn Labortechnik) for tensile strain in DMEM High Glucose (D5671, Sigma Aldrich), supplemented with 10% FBS (P30-3302, PAN Biotech), 1% AA (A5955, Sigma Aldrich), 1% L-glutamine (G7513, Sigma Aldrich) and 1% ascorbic acid (B6891, Sigma Aldrich). We either left the cells untreated or added 200 µl A. actinomycetemcomitans (Agac) lysate for 24 h [23 (link)]. After that we compressed PDLF with a sterile glass plate (2 g/cm²) [24 (link)] or stretched them using a spherical silicone stamp (16%) [25 (link)] for another 48 h according to validated and published protocols without changing the cell culture medium.

The Huh7 and Hep3B cell lines were purchased from Shanghai Gene Chem (Shanghai, China). The Huh7 cell line was routinely cultured in DMEM medium (Biological Industries, Shanghai, China) containing 10% fetal bovine serum (FBS) (P30–3302; PAN Biotech), 100 U/mL penicillin and 100 μg/mL streptomycin. The Hep3B cell lines was cultured in minimum essential medium (MEM) (Gibco, Shanghai, China) containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37 °C under a 5% CO₂ atmosphere.

HEK-293T cells were grown in DMEM (SH30022, Hyclone/GE, United States) supplemented with 10% fetal bovine serum (P30-3302, PAN-Biotech, Germany) plus 100 IU/mL penicillin and 0.1 mg/mL streptomycin (SV30010, Hyclone/GE, United States) at 37 °C, 5% CO₂. Approximately 3 × 10⁵ cells were seeded in each well of a 6-well plate 1 day before transfection. When cells reached 70%~90% confluency, 100 ng of the firefly luciferase reporter pP_PK2::Fluc, 40 ng of the renilla luciferase reporterpP_CMV::Rluc, 250 ng of a hamster Bmal1 construct, and 250 ng of a mouse Clock construct and 500 ng of mouse Per2 (or human CHR, CHR-N, CHRNH, CHR-H, CHRH2, CHRH3, CHRH4, CHRHC, and CHR-C) were co-transfected into HEK 293T by using Lipo6000™ Transfection Reagent (C0526, Beyotime, Beijing, China). The constant amount of DNA (1100 ng/well) was adjusted with pcDNA3.1(+)/Hygro vector as a carrier, and we measured single luminescence. Twenty-four hours after transfection, cells were washed three times with ice-cold PBS (SH30256.01, Hyclone/GE, United States), then processed with a TransDetect Double-Luciferase Reporter Assay Kit (FR201, Transgen Biotech, Beijing, China), and measured by SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, Silicon Valley, United States). Each construct was examined at least for three independent times.

To assess the optimal compression time in the POR scaffold, PDLFs were cultured for 4, 24, 48, and 72 h either on conventional 24-well cell culture plates (662-160, Greiner Bio-One GmbH, Frickenhausen, Germany) or on 24-well INO matrix plates (Porous POR PCL Morphology Single Type Plate, 811004-24, Biozym Scientific GmbH, Hessisch Oldendorf, Germany) in DMEM High Glucose (D5671, Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS (P30-3302, PAN-Biotech GmbH, Aidenbach, Germany); 1% antibiotic/antimycotic (A5955, Merck KGaA, Darmstadt, Germany); 1% L-glutamine (G7513, Merck KGaA, Darmstadt, Germany), and 100 µM ascorbic acid (A8960, Merck KGaA, Darmstadt, Germany). After 24 h of preincubation, the cells were either left untreated or compressed with ZnO₂ plates (2 g/cm²). Depending on the periods to be investigated, the plates were left on the cells for 4, 24, 48, or 72 h. After the corresponding incubation time, RNA was isolated and analysed with RT–qPCR. The supernatant was used for the LDH assay (Supplemental Materials).

The human HNSCC cell lines, CAL27, HN30, HN6, FADU, and human embryonic kidney cell line, 293 T were cultured in DMEM high glucose media (SH30022.01, cytiva, China) with 10% fetal bovine serum (P30-3302, PAN-Biotech, German). All cells were cultured in a humidified incubator (Forma™ 351, Thermo Fisher) at 37 °C with 5% CO₂.