Flexstation 3

Antibody internalization assay was performed as described previously [45 (link),46 (link)] with minor modifications. Briefly, cells at the concentration of 5000 per ml were seeded onto 96-well plates. After 24 h, control IgG or SEMA6C antibody was added. One hour post-antibody addition, cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) for 10 min, and permeabilized with 0.05% Tween 20 PBS for 10 min. An internalized antibody was detected by a polymer-conjugated secondary antibody (NICHIREI, Tokyo, Japan) and then by 3, 3′, 5, 5′-tetramethylbenzidine (TMB; Sigma). The reaction was stopped by 2N H₂SO₄, and the end product was measured at O.D. 450 nm by FlexStation 3 (Molecular Probes; Waltham, MA, USA).

NHDF were seeded at 20,000 cells/well in duplicate in 24 well plates in FGM-2 media the day before treatment. Media was changed to DMEM + 10% FBS and stimulated with PALA (1–25 µM) for 1 h prior to infection with SAP227 (MOI = 5) for 2 hours. Bioluminescence of SAP227 was monitored at 30 minute intervals for 10 s/well in a Flexstation 3 (Molecular Probes, Eugene; OR). After 2 h, supernatants were collected and cells lysed in 50 µL 1%Triton X-100/PBS on ice for 10 minutes. Cell lysates and supernatants were combined and viable bacteria determined through serial dilution and plating of duplicate samples on LB agar plates.

FRET time-course of cAMP intracellular levels was measured as previously described (Carozzo et al., 2019 (link)). Briefly, HEKT Epac-S^H187 transfected with pCDNA3.1 HA-β3-adrenergic receptor alone or with pCEFL GRK2 pC were seeded in 96-well plates at a density of 1 x 10⁵ cells/well. Before starting each experiment, cells were washed with NaCl 0.9% twice and 100 μl of FluoroBrite DMEM (Thermofisher) was added to each well before placing the plate in a FlexStation^® 3 at 37°C. In order to determine intracellular-cAMP (i-cAMP) response, we measured the baseline fluorescence signal detected at 475 nm (donor) and 530 nm (FRET) emission with excitation at 430 nm. Using the on-board pipettor, we added 50 μl of 3X BRL37344 after 40 s and then monitored the signal every 20 s for a total of 600 s. Different concentrations of BRL37344 were assayed for each transfection. FRET and donor intensities were measured for each time point. FRET/donor ratio was calculated and normalized to basal levels -before stimulation- (R/R₀) for each time point. An AUC value of 10-minute R/R₀ i-cAMP response was calculated for each replicate. Concentration-response curves were constructed plotting AUC values of 10-minute R/R₀ i-cAMP response vs log [BRL37344].

Measuring the intracellular Ca²⁺ level in CHO-K1 cells containing GRP10 was performed using the calcium-sensitive dye Fura-2 according to the manufacturer’s protocol (Molecular Devices, Sunnyvale, CA, USA). The day before the experiment, cells were seeded at 40,000 cells/well in 96-well plates in growth media and kept at 37 °C in an incubator with 5% CO₂ overnight. Peptides were tested at concentrations from 10⁻¹² to 10⁻⁵ M. Fura-2 fluorescent dye was detected using a FlexStation 3 fluorometric plate reader (Molecular Devices), and excitation was measured at 340 nm and 380 nm and emission at 510 nm.
The intracellular Ca²⁺ level was measured using the AequoScreen stable CHO-K1 cell line containing NPFF-R2 purchased from Perkin Elmer according to the manufacturer’s protocol. Cells at 80–90% confluence cultured in media without selective antibiotics were detached (PBS pH 7.4 + 0.5 mM EDTA) and centrifuged. Cells resuspended in phenol red-free DMEM with 0.1% protease-free BSA and 5 µM coelenterazine h (Thermo Fisher Scientific Inc. Brand) were seeded at 50,000 cells/well in 96-well plates and incubated in the dark at RT with gentle agitation for 4 h. Peptides were tested at concentrations from 10⁻¹² to 10⁻⁵ M. Luminescent light emission was recorded using a FlexStation 3 plate reader.

Cell Counting Kit‐8 Method: Cell Counting Kit (Beyotime, China) was applied to evaluate the cytotoxicity of the material itself on BMSCs. Cells were seeded in 96‐well plates at a density of 0.5 × 10⁴ mL⁻¹ and added with MHS (0.1 mg mL⁻¹), MHS @ PPKHF (0.1 mg mL⁻¹), PPHF (1 µm), and PPKHF (1 µm), respectively. The plates were placed in an incubator at 37 °C in a 5% CO² humidified atmosphere and the medium was changed every 2 days. 90 µL of DMEM medium and 10 µL of CCK‐8 were added to each well at 12, 24, and 48 h and the absorbance was detected at 450 nm using a microplate reader (FlexStation3, Japan) after further incubation for 2 h.
live/dead staining Experiment: Cell viability of BMSCs was determined by live/dead staining (Life Technologies). Live cells were stained with calcein AM and dead cells were stained with PI. In short, BMSCs were seeded at a density of 5 × 10⁴ per mL in 24‐well plates and added with MHS (0.1 mg mL⁻¹), MHS @ PPKHF (0.1 mg mL⁻¹), PPHF (1 µm), and PPKHF (1 µm), respectively. After incubation for 12, 24, and 48 h at 37 °C in 5% CO₂ atmosphere, the culture medium was aspirated, and the cells were thoroughly washed with PBS before adding the dye solution to each well. After incubation for 40 min at room temperature, the cells were detected by fluorescence microscopy (OLYMPUS, SONY, Japan).