Tissuelyser lt

Colon samples of around 1.5 cm were homogenized in 300 µl of TRIzol reagent (Thermo Fisher Scientific, USA) in the TissueLyser LT (QUIAGEN, Hilden, Germany) for 5 min at 50 s⁻¹ and 4 °C. Total cytoplasmic RNA was prepared from samples following the TRIzol datasheet; aliquots were converted to complementary DNA (cDNA) by reverse transcription using random hexamer primers. Semi-quantitative changes in messenger RNA (mRNA) levels were estimated by RT-qPCR using specific conditions: 35–40 cycles of denaturation at 95 °C for 10 s, annealing at 58–65 °C for 15 s depending on the specific set of primers, and extension at 72 °C for 20 s. Reactions were carried out in presence of SYBR green Quantimix Easy Master Mix (Biotools Biotechnological & Medical Laboratories SA Labs, S.A., Madrid, Spain) in a 20 μl reaction in a Rotor-Gene (Corbett Research, Mortlake, NSW, Australia). Primer oligonucleotides for PCR were designed with the Primer3 tool. Target specificity was checked by in silico PCR using the USCS GenomeBrowser and Blast (NCBI) for cDNA and genomic DNA; only primer pairs with no unintended targets were selected (see Supplementary Table S1 online). Relative mRNA concentrations were calculated from the take-off point of reactions using the included software, and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) levels were used as a housekeeper.

To analyze the early gene response of hMSC to mechanical stimuli, two scaffolds for each condition were harvested in Trizol (Sigma), and cells were lysed for 5 min at 50 Hz (TissueLyser LT, Quiagen, Hilden, Germany). RNA was isolated by 1-bromo-3-chloropropane (B9673, Sigma-Aldrich) phase separation followed by column separation according to the manufacturer’s instructions (NucleoSpin RNA, Macherey-Nagel, Dueren, Germany). cDNA was synthesized by reverse transcription (Promega, Walldorf, Germany) from 1 µg of RNA.
Real-time Polymerase chain reaction (PCR) was performed by CFX96 Real-Time System (Bio-Rad). Primers for genes targeting either mechanosensory functions—fos proto-oncogen (cFos), prostaglandin-endoperoxide synthase 2 (Cox2), integrin subunit beta 5 (ITGb5), osteopontin (SPP1), bone morphogenetic protein 2 (BMP-2)—or osteochondral early differentiation—collagen type-VI (Col6), runt-related transcription factor 2 (Runx2), SRY-Box Transcription Factor 9 (Sox9)—were designed in the Primer Blast tool from NBCI and purchased from Biomers.net (Ulm, Germany). Primers sequence and NCBI reference numbers appear in Supplementary Table S1.
Expression of target genes was normalized with beta-2-microglobulin (B2M, NM_004048.2) as the reference gene, and results displayed as relative values (10,000 × 2^−ΔCt).

Monoaminergic neurochemicals were extracted from 8 pools of 5 whole-larvae, 8 pools of 15 heads, and 8 pools of 15 trunks following an extraction procedure adapted from Mayol-Cabré et al. (2020) [27 (link)]. The extraction process was based on the use of a solvent of polarity similar enough to the neurotransmitters to be able to extract them from the sample. First, the samples with the extractant solvent were homogenized using stainless steel beads in a mill homogenizer (TissueLyser LT, Quiagen, Hilden, Germany). Then, the resulting supernatant was centrifuged and filtered and introduced into a chromatographic vial. The analysis was performed by ultra-high-performance liquid chromatography (Acquity UPLCH-Class Waters, Milford, MA, USA) coupled to a triple quadrupole mass spectrometer (Xevo, TQS micro, Waters, Milford, MA, USA) equipped with an electrospray ionization source (ESI). Total protein of the samples was measured by the Bradford method [28 (link)] using bovine serum albumin (BSA) as the standard. Results were normalized by two different methods, by larva and by protein content. Additional details on the extraction and analysis of neurotransmitters are provided in the Supplementary Methods and Supplementary Table S1.

Brain samples were homogenated and lysed with RIPA buffer (10⁻² M Tris-Cl (pH 8.0), 10⁻³ M EDTA, 5 × 10⁻⁴ M EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.14 × 10⁻³ M NaCl, 1 × protease inhibitor cocktail (#P8340), 1 × phosphatase inhibitor cocktail 2 and 3 (#P5726, P0044), 10⁻³ M PMSF (all from Sigma-Aldrich, St. Louis, MO, USA) using Tissue LyserLT (Quiagen, Hombrechtikon, Switzerland). Protein concentration was determined using BCA Protein Assay Kit (#23225, Thermo Fisher Scientific, Waltham, MA, USA). Lysates were loaded on SDS-PAGE, and separated proteins were transferred onto the nitrocellulose membranes (#GE10600002, Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked in 5% not-fat milk or 5% BSA in TBS-T for 60 min at room temperature, incubated with the appropriate primary antibody (+4 °C, overnight) and then with a secondary antibody conjugated with HRP (60 min at room temperature). Bands were visualized using SignalFire Plus ECL Reagent (#12630, Cell Signaling Technology, Danvers, MA, USA). Membranes were scanned using Amersham Imager 680 (GE HealthCare, Chicago, IL, USA) and quantified in the Image Quant TL v.8.1 (GE HealthCare, Chicago, IL, USA). Measurement of each protein was then normalized on the related α-Tubulin loading control.

During batch culture using glucose as the sole carbon source, C. acetobutylicum cells were collected at 8, 16 and 24 h by centrifugation at 10,000×g for 3 min at − 10 °C. The resulting cell pellets were quenched immediately with 500 μL solution mixture of methanol, acetonitrile and water (40:40:20, v/v, − 40 °C), and then frozen in liquid nitrogen for preparing crude extracts. According to our previous study [22 (link)], LC–MS/MS analysis was conducted for ATP quantification with an ACCELA HPLC system (Thermo Scientific, CA) equipped with an XBridge BEH Amide column (100 mm × 2.1 mm I.D., 2.5 μm, Waters, Ireland). Mass monitoring was achieved using a TSQ Quantum Ultra triple quadrupole mass analyzer (Thermo Scientific, CA) equipped with a heated electrospray ionization source (HESI). NADH assay was performed using a commercial kit (Sigma, MO). Cell pellets were first lysed using a Qiagen Tissue Lyser LT (Qiagen, Germany) at 50 oscillations/s for 3 min in the NADH extraction buffers (Sigma, MO), the resulting lysate was then used for NADH quantification at 450 nm with an iMark™ microplate reader (Bio-Rad, CA).

Total RNA was harvested from the livers using a Qiagen Tissue Lyser LT (Qiagen) and then extracted using the RNeasy kit (Qiagen). We quantitated the total RNA on a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR amplification of the cDNA was performed by quantitative real-time PCR for gene-specific primers as previously described using TrueAmp SYBR Green qPCR SuperMix (Alkali Scientific). The thermocycling protocol consisted of 5 min at 95 °C, 40 cycles of 15 s at 95 °C, and 30 s at 60 °C, finished with a melting curve ranging from 60 to 95 °C to allow the distinction of specific products. Normalization was performed in separate reactions with primers to 36B4.