Proteinase k

Polyacrylamide droplets (200 μl) were suspended in 800 μl of 1x PCR buffer (Fermentas), after which 13 U of proteinase K (Sigma–Aldrich, St. Louis, MO, USA) was added and the suspension was incubated at 37°C for 1 h. After heat-inactivating proteinase K at 90°C for 1 min, 20 μg of lysozyme (Sigma, St. Louis, MO, USA) was added and the suspension was incubated at 37°C for 1 h. Finally, 26 U of proteinase K was added and the suspension was incubated for 5 h at 55°C. Enzymes were inactivated by heating at 90°C for 5 min.

The isolated BMSC-EVs were incubated with proteinase K (0.05 μg/μL; Sigma Aldrich) for 10 min at 37°C and then with 5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma Aldrich) for 10 min at ambient temperature to limit proteinase K activity, followed by complete inactivation of proteinase K by heating at 90°C for 5 min. Following this, samples were incubated with RNase A at a final concentration of 0.5 μg/μL (Thermo Fisher Scientific) for 20 min at 37°C to digest the exposed RNA. In the control group, proteinase K, PMSF, or RNase A was substituted by the same amount of PBS. RNA was finally extracted and used for subsequent analysis.

Extracts from A375 or A2058 cells were used for RIP assay using the EZ-Magna RIP kits (17–701, Millipore, Billerica, MA, USA). A portion of supernatant was used as Input. The other portion of supernatant was incubated with 1 mg magnetic beads precoated with antibodies against IgG (ab172730, Abcam, Cambridge, MA, USA) or HuR (ab200342, Abcam) at 4˚C overnight. Next, the RNA compound was incubated with proteinase K (RIP washing buffer, 10% sodium dodecyl sulfate (SDS), 10 mg/mL proteinase K, Millipore) at 55°C for 30 min to digest the remaining protein on the beads. Afterwards, immunoprecipitated RNA was purified and analyzed by RT-qPCR.

Mitochondrial and cytoplasmic fractions were extracted using the Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, #89874, Waltham, MA, USA). Briefly, cells were collected and then incubated with Reagent A for 2 min on ice. Reagent B was added, and the sample was vortexed at maximum speed, followed by incubation for 5 min on ice. Reagent C was added, and the sample was centrifuged at 700× g at 4 °C for 10 min. The supernatant was collected and centrifuged at 12,000× g at 4 °C for 15 min. The supernatant represented the cytoplasmic fraction, and the pellet represented the mitochondria-enriched fraction. The pellet was washed using Reagent C and then lysed with SDS lysis buffer. For digestion with proteinase K, equivalent weights of the mitochondria-enriched fraction were resuspended in digestion buffer (250 mM sucrose, 0.5 mM EGTA, 0.5 mM EDTA, 3 mM HEPES-NaOH, pH 7.2), and proteinase K (Sigma-Aldrich) was added to a final concentration of 0.25, 0.5, 1, or 2 μg/mL with or without 1% (v/v) Triton X-100, with incubation for 20 min on ice. The reaction was stopped by the addition of 2 mM phenylmethylsulfonyl fluoride, and samples were collected and lysed after centrifugation at 15,000× g at 4 °C for 10 min.

Suspensions were prepared from individual colonies after culture on Blood Columbia agar. OD at 600 nm was measured and suspensions were diluted to 10⁹ CFU/ml in EDTA-saline buffer (75 mmol/L NaCl and 25 mmol/L EDTA, pH 7.5), then mixed with an equal volume of 1% low-melting-point agarose and allowed to solidify in a 100 μL plug mould. The agarose plug was incubated for 24 h at 37°C in 500 μL lysis buffer (6 mmol/L Tris–HCl (pH 7.6), 0.1 mol/L EDTA, 1 mol/L NaCl, 0.5% Brij®58 (polyoxyethylene (20) cetyl ether; Sigma), 0.4% sodium deoxycholate, 0.5% sodium lauryl sarcosine and 1 mg/mL lysozyme (and 10 µg/mL lysostaphin to S. aureus)). The lysis buffer was replaced with 500 μL proteinase K buffer (1% sodium lauryl sarcosine, 0.5 mol/L EDTA (pH 9) and proteinase K (50 μg/mL; Sigma)) and this solution was incubated with gentle shaking at 50°C for 20 h. The plugs were then washed four times for 30 min at 37°C with 10 mL of Tris–EDTA buffer (10 mmol/L Tris–HCl (pH 8) and 1 mmol/L EDTA). One-third of a slice of each plug was cut and incubated for 18–20 h with 30 U of SpeI, or SmaI, XbaI and Cfr9I in the restriction buffer (Thermo scientific). DNA restriction fragments were separated in a PFGE apparatus at 14°C, 6 V/cm, for a specific time for each species (19–23 h). The gel was stained with gel-red and visualized with a UV system.

Approximately 84 μL of the sample (70 μg of the extracted flagellar protein) was made up to 100 μL in 100 mM of ammonium bicarbonate (Fluka Analytical, UK) and denatured at 95°C for 5 min. Proteinase K (Sigma Aldrich, Dorset, UK) (1 μg/μL in 50 mM Tris‐HCl (Fisher Scientific, Loughborough, UK), 5 mM CaCl₂ (BHD laboratory, England)) was added to the mixture to give a 1:1 (replicate#1) or 1:100 (replicate#2) ratio of enzyme to protein, and incubated for 1 h (replicate#1) or 2 h (replicate#2) at 37°C with mild shaking. Proteolysis was quenched by freezing at –20°C and the sample stored until further analysis.
Both trypsin‐ and Proteinase K‐treated samples were desalted using C₁₈ column zip tips (Merck Millipore Ltd., Germany) (according to manufacturers’ instructions). Briefly, the tips were wetted using 100% acetonitrile (J.T., Baker, Holland), and then equilibrated using 0.1% trifluoroacetic acid (Fisher Scientific Loughborough, UK). The samples were aspirated and dispensed for 10 cycles, and then washed three times using 0.1% TFA and eluted using 70:30 acetonitrile and 0.1% TFA. The desalted samples were dried and re‐suspended in 10 μL of 0.1% formic acid prior to MS analysis.

Pregnant dams were euthanized with i.p. injection of ketamine (0.8 mL/kg, Narketan; Vetoquinol, Bern, Switzerland) and xylazine (0.6 mL/kg, Xylapan; Vetoquinol, Lure. Cedex, France) on GD 15 or GD 20. Placentas were isolated, weighted (all 352), and fixed in St. Marie solution (96% EtOH with 1% glacial acetic acid), dehydrated, and embedded in paraffin. Serial sections (5 µm) were cut for histology or immunohistochemistry on a Leica microtome.
DNA was isolated in TE buffer pH9 with 0.1 mg/mL of Proteinase K and 0.25% of Nonidet P40 (both from (Sigma, St. Louis, MO, USA) at 56 °C for 24 h, heated for 10 min at 95 °C to inactivate Proteinase K, spun and the supernatant was then frozen at −20 °C. DNA concentration and quality were measured with the NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, NC, USA).