Fetal bovine serum (fbs)

The MCF-7, HCC1937, MDA-MB-231, MDA-MB-468, MDA-MB-453, and SK-BR-3 cell lines were acquired from American Type Culture Collection (Virginia, USA). The MDA-MB-468 with STAT3 overexpression cell line was generously provided by Dr. Liu CY, working in Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital (Taipei, Taiwan). All cell lines were immediately expanded and frozen down immediately after acquiring. All cell lines could be restarted every 3 months from a frozen vial of the same batch of cells. Cells except for MDA-MB-468 with STAT3 overexpression were maintained as described culture medium by ATCC; MDA-MB-468 with STAT3 overexpression cells was maintained in L-15 medium with G418 700 μg/mL. All media were supplemented with 10% FBS (Caisson, USA), 100 units/mL penicillin, 100 mg/mL streptomycin, and 25 mg/mL amphotericin B (Caisson, USA). All human breast cancer cell lines were incubated in a humidified incubator at 37°C in an atmosphere of 5% CO₂ in air.

Canine CMT cell lines CMT-1 and MPG were kindly provided by Dr. Lin CT of the School of Veterinary Medicine, National Taiwan University (Taipei, Taiwan). Both were cultured in Dulbecco's modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Caisson) and 1% penicillin/streptomycin (Caisson) at 37°C in a humidified atmosphere of 5% CO₂. To verify the role of KMO in cell growth, 3000 cells/well of CMT-1 or MPG cells were seeded in a 96-well plate and treated with KMO inhibitor, Ro 61-8048 (Sigma-Aldrich), at the indicated concentrations for 24, 48, and 72 h. After the treatment, quantification of cell proliferation was performed using WST-1 reagent according to the manufacturer's protocol (Roche). For KMO knockdown, small interfering RNAs (siRNAs), including control and KMO, were used, and the reagents were all purchased from Santa Cruz Biotech Inc. CMT-1 and MPG cells were transfected for 48 h with siRNAs against KMO (Forward 5′-CCAAGGUAUUCCCAUGAGATT-3′, reverse 5′-UCUCAUGGGAAUACCUUGGTT-3′; scramble siRNA duplex: forward: 5′-UUCUC CGAACGUGUCACGUTT-3′; reverse: 5′-ACGUGACACGUUCGGAGAATT-3′). The cell viability of the cells was quantified using WST-1 and cell extracts were analyzed by KMO immunoblotting.

To investigate which components are needed to induce motility of spz, the mosquitoes were dissected and the obtained salivary glands were crushed in eleven different formulations: (1) Phosphate buffered saline which contains potassium, sodium, chloride and phosphate (PBS; Life Technologies Inc.), (2) Hanks’ balanced salt solution which contains besides the salts present in PBS also glucose, calcium, magnesium, sulphate and bicarbonate (HBSS; Life Technologies Inc.), (3) Roswell Park Memorial Institute medium (RPMI; Life Technologies Inc.) which contains besides the components of HBSS also amino acids and vitamins, (4) PBS enriched with 3.5 mg/ml bovine serum albumin (BSA; Sigma-Aldrich), (5) HBSS + 3.5 mg/ml BSA, (6) RPMI + 3.5 mg/ml BSA, (7) PBS enriched with 10% fetal bovine serum (FBS; Life Technologies Inc.), (8) HBSS + 10% FBS, (9) RPMI + 10% FBS, (10) RPMI without amino acids (Caisson Labs) + 3.5 mg/ml BSA and (11) RPMI without glucose (Life Technologies Inc.) + 3 mg/ml BSA. All eleven formulations had a physiological pH of 7.0–7.5 and a viscosity of < 1.1 cP. For imaging of the spz, 10 µl of the spz solution was pipetted on the cover slip of a confocal dish without any precoating (ø14 mm; MatTek Corporation), covered with another cover slip (ø12 mm; VWR Avantor) and imaged within 45 min. The set-up is schematically depicted in Additional file 1: Fig. S1.

Mouse neural stem cells (NSCs) were used for the cell culture test. NSCs derived from the brains of adult mice were cultured in Ham’s F-12 and HG-DMEM (1:1), with 10% fetal bovine serum (FBS; Caisson), 1% penicillin-streptomycin-amphotericin (PSA; Caisson), and 400 mg/mL G418 (Invitrogen), in a humid incubator containing 5% CO₂ at 37 °C. The culture medium was refreshed every day. The recombinant spider silk-coated surface (area of 1.9 cm²), in a 24-well plate, was immersed in 75% ethanol for 1 h and irradiated with UV light for 1 h before NSC inoculation (cell density 4 × 10⁴ cells/cm²). The proliferation of cells on the surface was evaluated by the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich) assay [27 (link)]. The reacted CCK-8 solution was mixed gently to ensure uniformity and then collected into a 96-well plate. The absorbance, at a wavelength of 450 nm, was detected by a plate reader (SpectraMax M5, Molecular Devices, San Jose, CA, USA). Statistical differences between the experimental groups were performed with the student’s t-test. The result was considered statistically significant when the p-value was smaller than 0.05.

Mouse 3T3-L1 fibroblast and RAW264.7 macrophage were kindly provided by Dr. Lin of the National Taiwan University and Dr. Tsai of the National Taiwan Normal University (Taipei, Taiwan), respectively. The original cell lines were purchased from Bioresource Collection and Research Center, Food Industry Research and Development Institute (Hsinchu, Taiwan). Mouse 3T3-L1 and RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Caisson, Smithfield, UT, USA) containing 10% heat-inactivated calf serum (BS, Gibco, Grand Island, NY, USA) and fetal bovine serum (FBS, Genedirex, Las Vegas, NV, USA), respectively. Murine 4T1 breast cancer cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in DMEM containing 10% heat-inactivated FBS with 1% penicillin/streptomycin/amphotericin B (Caisson) at 37°C in an incubator containing a humidified atmosphere of 5% CO₂. Aspirin was purchased from Sigma (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO, Sigma) as a stock solution, and then stored at −20°C. The concentration of DMSO in the vehicle group was equal to the 0.25% DMSO in the highest (5 mM) dose of Aspirin.