Fastprep 24 device

Portions (~30 mg) of the RNA later-stored tissues were homogenized using the FastPrep-24 device (MP Biochemicals, Irvine, CA, USA). RNA was extracted from the homogenates using the Nucleospin RNA II kit (Macherey-Nagel, Düren, Germany) essentially according to the manufacturer’s instructions. One µg RNA of each sample was reversely transcribed for 30 min at 50 °C into cDNA by means of Maxima Reverse Transcriptase (Thermo Fisher Scientific). Target gene-specific sequences were quantified proportionately to glycerin aldehyde-3-phosphate dehydrogenase (GAPDH) by real-time PCR using TaqMan Gene Expression Assays (ABI; Thermo Fisher Scientific). The reported relative gene expression data were calculated by relating the obtained qPCR data of corresponding tumor bearing and not tumor bearing sections by the ΔΔC_T method.

Starting from the control period, samples for microbial community analysis were collected once per week from each colon vessel. DNA was isolated using a previously described method [43 (link)], with some minor modifications. Luminal DNA was extracted from pelleted bacterial cells obtained from a 1 mL sample, while mucosal DNA originated from 0.25 g mucin agar collected from the mucin beads. Homogenization was performed using a Fastprep-24 device (MP BioMedicals, Illkirch, France) performed twice for 40 s at 4 m/s with a resting period of 5 min between shakings.
Subsequently, quantitative polymerase chain reaction (qPCR) for the Firmicutes phylum, the Bacteroidetes phylum, Akkermansia muciniphila, Bifidobacterium spp. and Lactobacillus spp. was performed on a QuantStudio 5 Real-Time PCR system (Applied Biosystems, Foster City, CA USA). Each sample was analysed in technical triplicate and outliers (more than 1 C_T difference) were omitted. Different published qPCR methods were adopted for the Firmicutes and Bacteroidetes phyla [44 (link)], Akkermansia muciniphila [45 (link)], and Lactobacillus and Bifidobacterium spp [46 (link), 47 (link)].

Frozen yeast cell pellets were resuspended in lysis buffer (8 M urea, 50 mM Tris buffer pH 8.0, 150 mM NaCl, 1 mM PMSF, 5 mM sodium butyrate, benzonase, and protease inhibitor cocktail) and subsequently lysed by bead-beating (FastPrep-24 device, MP Biomedicals). Frozen Jurkat E6-1 cells were dissolved in lysis buffer and lysed via sonication. After removal of insoluble debris by centrifugation at 16,000g at 4 °C for 10 min, yeast and human proteins were precipitated by the addition of three times the volume of cold acetone and subsequently incubated overnight at −20 °C. Proteins were pelleted by centrifugation at 15,000g, dissolved in 8 M urea, 50 mM ammonium bicarbonate buffer, and treated with 10 mM DTT for 45 min to reduce protein disulfide bonds. Alkylation of reduced cysteines was performed by adding iodoacetamide to a final concentration of 20 mM, followed by incubation for 30 min in the dark. The remaining iodoacetamide was quenched, and samples were diluted to 4 M urea for digestion with Lys-C 1:50 (w/w). After incubation for 2 h at room temperature, samples were further diluted to a final concentration of 1 M urea, and digestion with trypsin 1:50 (w/w) was carried out overnight at 37 °C. Finally, tryptic peptides were desalted using SepPak C18 cartridges (Waters), and eluates were shortly vacuum centrifuged and subsequently lyophilized.

The JPC-seeded scaffolds were placed in Lysing Matrix D microtubes with ceramic beads (MP Biomedicals, Irvine, CA, USA) and lysis buffer (Macherey-Nagel, Dueren, Germany), and shredded by a FastPrep-24 device (MP Biomedicals, Irvine, CA, USA). RNA isolation from the obtained lysates of JPC-seeded scaffolds was carried out using the NucleoSpin RNA Mini kit (Macherey-Nagel, Dueren, Germany) following the manufacturer’s instructions. After RNA quantification using the Nanodrop spectral photometer (Thermo Fisher Scientific, Waltham, USA), 200 ng of RNA was synthesized to cDNA using the SuperScript VILO kit (Thermo Fisher, Darmstadt, Germany) following the manufacturer’s instructions. mRNA transcription levels were quantified by a real-time LightCycler system (Roche Diagnostics, Mannheim, Germany). DNA Master SYBR Green 1 (Roche, Mannheim, Germany) and the primer kits (Search LC, Heidelberg, Germany) for the target genes (ALPL, COL1A1, RUNX2, OCN, GAPDH) were used for the PCR reactions. 35 cycles of amplification were carried out for each mRNA. The ratio of target gene copy numbers to those of the housekeeping gene (GAPDH) were calculated.

Mycelial samples were separated from the agar cultures with a sterile blade, frozen at -20°C and freeze-dried for 24 h in a VirTis SP Scientific sentry 2.0 equipment (SP Industries, Warminster, PA, USA). The dry samples were weighed and disrupted in a FAST-PREP 24 device (MP biomedicals, Irvine, CA) or in a Precellys 24 homogenizer (Bertin Technologies, Montigny le Bretonneux, France) with 1 ml of acetone as described [57 (link)]. Extractions were repeated up to total bleaching of the samples (3–6 extractions depending on carotenoid content) and the extracted solvent was dried in a Concentrator Plus equipment (Eppendorf, Hamburg, Germany). Dry samples were resuspended either in 1 ml or 0.1 ml hexane, depending on carotenoid concentrations, and subjected to spectrophotometrical determinations in the range of 350–650 nm (Shimadzu UV spectrophotometer 1800). Maximal absorbance at 482 nm was used to estimate the total amount of carotenoids, based on an average maximal E (1 mg l^-1, 1 cm) of 200, and normalized according to the dried weight and dilution of the sample. When required (i.e., in analyses of ΔcryD or ΔwcoA mutants), bikaverins were removed from the carotenoid fraction as described [27 (link)].

Each compartment dissected was homogenized individually using a FastPrep-24 device (MP Biomedicals, Illkirch-Graffenstaden, France) and glass beads (Sigma, Lyon, France) in a mix (50/50) of 15 μL 70% (v/v) formic acid (Sigma) and 15 μL 50% (v/v) acetonitrile (Fluka, Buchs, Switzerland) for protein extraction according to the standardized automated setting described by Nebbak et al. [22 (link)]. A quick spin centrifugation at 200 g for 1 min was then performed and 1 μL of the supernatant of each sample was spotted on the MALDI-TOF steel target plate in quadruplicate (Bruker Daltonics, Wissembourg, France). After air-drying, 1 μL of matrix solution composed of saturated α-cyano-4-hydroxycinnamic acid (Sigma, Lyon, France), 50% (v/v) acetonitrile, 2.5% (v/v) trifluoroacetic acid (Aldrich, Dorset, UK) and HPLC-grade water was added. Matrix solution was loaded in duplicate onto each MALDI-TOF plate with and without bacterial control (Pseudomonas aeruginosa ATCC 27853) respectively as a positive or negative control. Spectra were acquired on a Microflex LT MALDI-TOF Mass Spectrometer (Bruker Daltonics) as previously described [23 (link)].