Dithiothreitol (dtt)

Trophoblast cell cultures were rinsed twice with PBS before treatment with 0.25% trypsin for 5 min at 37 °C. The enzymatic reaction was stopped using 10% FBS/PBS. Cells were collected and filtered through a 70μm strainer for t0, t1, t4 and t24 treatments. For t36 and t48 treatments the cells were not filtered. Cells were centrifuged twice to remove traces of FBS, then counted and diluted to 200,000 cells/mL in PBS/0.1% BSA (A7906-100G, Sigma).
Cell suspensions were processed as per the Drop-seq protocol described by^{75 (link)}. In short: Microfluidic devices were manufactured using the Drop-seq 125um design in polydimethylsiloxane (PDMS) by replica moulding from a SU-8 master and plasma-bonded to glass slides. The device channels were rendered hydrophobic by flowing Aquapel through, flushing the excess and baking at 65 °C for 20 min. Single cells suspended in PBS/0.1% BSA and 120 primer-barcoded beads/µl (MACOSKO-2011-10, ChemGenes, Wilmington, USA) in 2x lysis buffer were loaded into 3 ml syringes. Using syringe pumps cells (4 ml/h) and beads (4 ml/h) were encapsulated in QX200™ Droplet Generation Oil for EvaGreen (#1864005, Biorad, Hertfordshire, UK) (15 ml/h) through a microfluidic device with a 125 µm channel. A magnetic stirrer was used in the bead suspension to avoid settling. The final concentration of lysis buffer achieved in the droplets was 3% Ficoll PM-400(F5415, Sigma), 0.1% Sarcosyl (L7414, Sigma), 0.01 M EDTA (AM9260G, Ambion), 0.1 M Tris pH 7.5 (15567027, Sigma) and 25 mM DTT (Sigma) which resulted in cell lysis and release of mRNA. Droplets were collected in a Falcon tube on ice for 15 min per run once stable droplet formation was achieved. After mRNA binding to the oligo-dT portion of the bead primer, the droplets were broken to pool beads for reverse transcription (EP0743, ThermoFisher). Exonuclease (EN0581, ThermoFisher) treatment removed unused primers from the beads prior to PCR amplification at up to 8,000 beads/tube ( ~ 400 cells/tube). All primer sequences used are detailed in the published Drop-Seq protocol and were purchased from IDT (Coralville, Iowa, USA).
Samples were pooled with up to 4,000 cells/sample and the DNA purified using Ampure XP beads (A63880, Beckman Coulter) before determining concentration with an Agilent HS DNA kit (5067-4626, Agilent, Stockport UK). DNA was fragmented and tagged with Illumina adapter and index sequences using Nextera Sequencing kit (FC-131-109, Illumina, Little Chesterford, UK) and 600 pg DNA/sample. Samples were diluted 1:1 in ddH₂0 water before double-sided purification with Ampure XP using 0.6 × beads to remove large fragments and 1 × beads twice to remove contaminating primers.
Samples were analysed for size distribution and concentration using an Agilent HS DNA kit and sequenced in multiple pools across multiple lanes of Illumina Nextseq, HiSeq2500 and HiSeq4000 flow cells due to institutional upgrades (Supplementary Data 23).
For the WT and Cxadr KO scRNAseq dataset three independently derived WT and Cxadr KO TSC clones each, grown in either stem cell conditions or differentiated for 24 h in Inhibit or Remove conditions, were used for scRNA-seq analysis using Evercode™ WT Mini v3 single cell whole transcriptome kit (Parse Bioscience).
Cells were fixed with the Evercode Cell Fixation v3 kit (Version 1.1, Parse Bioscience) following the manufacturer’s instructions. Cells were lifted and counted with an automated cell counter (Luna II, Logosbio). Approximately 1 million cells per sample were used for fixation. Cells were pelleted, then resuspended in prefixation master mix before straining into a new tube. Cells were incubated in fixative master mix and permeabilization buffer, followed by Perm Stop buffer, cell pelleting and resuspension in cell storage buffer. Cells were strained again, counted, then frozen and stored at -80 °C until further processing.
Fixed cells were processed with a Parse Evercode WT v3 kit as per the manufacturer’s instructions. Cells were counted and loaded using the Parse Biosciences Evercode WT Sample Loading Table v2 template into barcoding plates to yield 5000 barcoded cells per biological replicate. 8 sublibraries of 12,500 cells from all samples were generated after cell barcoding. All sublibraries were pooled and sequenced in one batch on 2 lanes of NovaSeq X 25B flowcell (R1:64, R2:58, I1:8, I2:8 with 10% PhiX) to yield average 60,000 reads per cell.

To prepare lysates, the cells were washed in ice-cold PBS and then extracted in ice-cold lysis buffer for 10 min (20 mM Tris–HCl (Sigma-Aldrich no. T4661) pH 7.4, 150 mM NaCl (Sigma-Aldrich no. S3014), 1% (v/v) IGEPAL (Sigma-Aldrich no. S3014), 0.1% (w/v) sodium deoxycholate (Sigma-Aldrich no. D6750), 40 mM sodium β-glycerophosphate (Sigma-Aldrich no. G9422), 10 mM NaF (Sigma-Aldrich no. 201154), 0.3 mM sodium orthovanadate (Sigma-Aldrich no. S6508), 100 nM okadaic acid (Enzo Life Sciences no. ALX-350-011-M001), 200 nM microcystin-LR (Enzo Life Sciences no. ALX-350-012-M001), 1 mM dithiothreitol (DTT) (Sigma-Aldrich no. 11583786001), protease inhibitor cocktail (Roche, cOmplete, EDTA-free protease inhibitor cocktail, no. 11873580001), and 1× phosphatase inhibitor cocktail (Sigma no. P0044) in water). The lysates were collected in 1.5 ml Eppendorf tubes (Eppendorf no. 10451043) and then clarified by centrifugation at 14,000 r.p.m. for 15 min at 4 °C. The protein concentrations were measured by Bradford assay using Protein Assay Dye Reagent Concentrate (Bio-Rad no. 5000006). The samples were normalized to 0.5–1 mg ml⁻¹. The sample buffer (0.1875 mM Tris–HCl (Sigma-Aldrich no. T4661) pH 6.8, 1% (w/v) SDS buffer (Sigma-Aldrich no. 75746), 10% (v/v) glycerol (Sigma-Aldrich no. G9012), 0.05% (w/v) bromophenol blue (Sigma-Aldrich no. B5525) and 10% (v/v) β-mercaptoethanol (Sigma-Aldrich no. M3148) in water) was added, and the samples were boiled at 95 °C for 10 min before progressing to SDS–polyacrylamide gel electrophoresis (PAGE) and western blotting.

Tectoridin was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China, Cat NO. 611-40-5) with a purity of 98.68%. TritonX-100, Tris, glycine, sodium dodecyl sulfate (SDS), Edaravone and dithiothreitol (DTT) were provided by Sigma-Aldrich, located in St. Louis, MO, USA. Madopar came from (Shanghai Roche Led, Shanghai, China). Primary antibodies against β-actin, Akt, p-Akt (Ser473), Bcl-2, Bax, mTOR, p-mTOR, TLR4, MyD88, NF-κB, Nrf2 and HO-1, horseradish peroxidase (HRP)-conjugated anti-rabbit, and HRP-conjugated rat antibodies were provided by Abcam (Cambridge, UK). All other chemicals were of analytical grade with high purity and were provided by Shanghai Chemical Reagent.

Protein samples in 25 μl SDS gel dissociation media were acidified by addition of 2 μl of 12% phosphoric acid then proteins were precipitated by addition of 200 μl 90% MeOH, 100 mM Triethylammonium bicarbonate buffer (TEAB, Honeywell Fluka cat# 60–044-974). Precipitation was initiated by incubating samples for one hour at 37°C and completed by overnight incubation at −20 C. Precipitates were pelleted by centrifugation for one minute at 16,000 x g, then washed by resuspending particulates in 400 μl of 90% MeOH, 10 mM TEAB. Precipitates were collected by centrifugation and dried on the bench then resuspended in 25 μl of 50 mM HEPES, pH 8.2. 0.25 μg Trypsin (Promega, V5113) was added to each sample and then incubated at 47°C for one hour then overnight at 37°C to complete the digestion. Following digestion, the samples were reduced and alkylated by incubating with 1 mM Dithiothretol (DTT, Sigma cat# D5545) for one hour at 37°C followed by addition of 3 mM Iodoacetamide (IAA, Sigma cat# I1149) for 30 minutes at room temperature in the dark followed by IAA quenching with addition of 1 mM DTT.

The 72 μL “eluate” samples were thawed for proteomic characterization. Analogous “input” samples were prepared by diluting aliquots of each input sample containing ~3.6 μg of protein (based upon BCA results) to 72 μL with 5 M Urea, 0.1% SDS, in PBS pH 7.40. Disulfide bonds were reduced with 5 mM dithiothreitol (DTT, Millipore Sigma) and heated to 37 °C for one hour; after cooling, samples were alkylated in the dark for 30 min using 30 mM iodoacetamide (Millipore Sigma). Alkylation was quenched with 5 mM DTT at room temperature for 15 min. Samples were prepared for mass spectrometry using Single-Pot, Solid-Phase-enhanced Sample Preparation (SP3) ^{[33 ]}. Briefly, samples were reconstituted by adding 100 μg of Sera-Mag^™ carboxylate-modified magnetic beads (50 μg/μL, Cytiva) to each sample, followed by an equal volume (90.6 μL) of absolute ethanol (Millipore Sigma). The samples were mixed at room temperature for 5 min on a thermomixer at 1,000 rpm to allow protein binding. Afterwards, samples were placed on a magnetic stand for 2 min and the supernatant was removed. Beads were washed three times by sequential resuspension in 180 μL of 80% ethanol, placing on a magnetic stand for 2 min, and removing the supernatant. Proteolytic digestion occurred on the beads after resuspension in 100 μL of 50 mM ammonium bicarbonate (ABC, Millipore Sigma) with 0.14 μg of trypsin (Promega, 1:25 ratio) for the inputs and dECM-tumor eluates and 0.072 μg of trypsin for the tumoroid eluates. The samples were digested for 2 hours at 47 °C, 1000 rpm. Following digestion, the samples were centrifuged at 20,000xg for 1 min at room temperature. The supernatant containing the tryptic peptides was removed, transferred to a new tube, and evaporated to dryness. Samples were reconstituted in 150 μL of 0.1% trifluoracetic acid (TFA) and cleaned up using a C18 ZipTip (Pierce, Thermo Fisher Scientific, 87784) according to the manufacturer’s instructions. After evaporation to dryness, samples were reconstituted with 8 μL of 5% acetonitrile containing 0.2% formic acid. Liquid chromatography-tandem mass spectrometry analysis was performed with 4 μL of each eluate sample and 2 μL of each input sample.
Samples were analyzed by a HPLC-MS/MS system consisting of a high-performance liquid chromatograph (HPLC, nanoAcquity, Waters) connected to an electrospray ionization (ESI) Orbitrap mass spectrometer (QE HF, Thermo Fisher Scientific). Injected peptides were loaded onto a 20 cm long fused silica capillary nano-column packed with C18 beads (1.7 μm in diameter, 130 Angstrom pore size from Waters BEH), with an emitter tip pulled approximately to 1 μm using a laser puller (Sutter instruments). Peptides were initially loaded on-column for 30 minutes at 400 nL/min at 98% buffer A (aqueous 0.1% formic acid) and 2% buffer B (acetonitrile with 0.1% formic acid) and then eluted over 120 min at a flow rate of 300 nL/min with a gradient as follows: time 1 min-2% buffer B; time 31 min-8% buffer B; time 111 min-44% buffer B; time 122–129 min-64% buffer B; time 133–152 min-equilibrate at 2% buffer B. The nano-column was held at 60 °C using a column heater constructed in house.
The nanospray source voltage was set to 2,200 V. Full-mass profile scans were performed in the FT-orbitrap between 375–1500 m/z at a resolution of 120,000, followed by MS/MS higher-energy collisional dissociation (HCD) scans of the ten highest intensity parent ions at 30% collision energy (CE) and 15,000 resolution, with a mass range starting at 100 m/z and a 2.5 m/z isolation window. Charge states 2–5 were included and dynamic exclusion was enabled with a repeat count of one over a duration of 15 s. The MS/MS orbitrap HCD scans were collected with automatic gain control (AGC) target set to 1e5 ions, a maximum inject time of 500 ms, and 1 microscan per spectrum.

The procedure for obtaining and extracting γ_B-crystallin has been described in detail elsewhere.^{17,27 (link)} Briefly, the filtered cellular contents of bovine eye-lenses were passed through a size exclusion chromatography column, yielding a mixture of γ-crystallins. This mixture was further separated using a fast flow ion exchange column where a salt gradient was used to elute the individual components with γ_B-crystallin being collected. The final sample was added to a 52.4 mM phosphate buffer corresponding to pH 7.1 in D₂O with the addition of 20 mM dl-dithiothreitol (DTT, Sigma Aldrich D9779), 1 mM ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA, Sigma Aldrich E5134) and 0.02 wt% sodium azide (NaN₃, Sigma Aldrich S2002). Amicon Ultra centrifugal filter units with a volume of 15 mL (cutoff 3 kDa) or 0.5 mL (cutoff 10 kDa) were used to concentrate the samples. The final protein volume fraction was determined from UV-absorption using an extinction coefficient (0.1%, 1 cm, 280 nm) of 2.18^{27 (link)} and converted to a volume fraction using a voluminosity of 0.71 mL g⁻¹.^{22 (link)}