Fraction collector

Wild-type (WT) and TAZ KO mouse embryonic fibroblasts (MEF) were seeded onto three 150-mm culture dishes at a density of 1.5 × 10⁶ cells and 3.75 × 10⁶ cells per dish, respectively. Two days after seeding, the MEF were washed with 10 mL of PBS and incubated in 100 μg/mL cycloheximide diluted in PBS at 37 °C for 20 min. After incubation, the cells were scraped, transferred to a conical tube, and centrifuged at 750 × g for 3 min. The supernatant was discarded and the pellets resuspended in 1 mL of lysis buffer (50 mM MOPS, 15 mM MgCl₂, 150 mM NaCl, 100 μg/mL cycloheximide, 0.5% Triton X-100, 1 mg/mL heparin, 0.2 U/μL RNase inhibitor, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamidine) and centrifuged. For fractionation, a sucrose gradient (10 mL of 10–50%) was established and the soluble fraction of the lysed sample was loaded onto the sucrose gradient and centrifuged at 160,000 × g at 4 °C for 2 h (SW-41 Ti rotor, Beckman Coulter, Brea, CA, USA). After centrifugation, the gradients were fractionated using an ISCO tube piercer (Brandel, Gaithersburg, MD, USA) and collected using a fraction collector (Bio-Rad, Hercules, CA, USA). cDNA was synthesized from total RNA isolated from each fraction using M-MLV reverse transcriptase and the mRNA of target genes was amplified by PCR. Amplicons were visualised and assessed by agarose gel electrophoresis.

Polysome profiles were obtained byt reating cells with 0.1 mg/mL cycloheximide (CHX) for 10 min at 37°C, washed twice with ice-cold PBS-CHX (phosphate buffered saline containing 0.1 mg/mL CHX), and harvested using polysome lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 0.4% IGEPAL, 50 μg/mL CHX, protease inhibitors, and RNaseIn). Lysates were clarified by centrifugation at 13,000 × g for 10 min at 4°C. Equal amounts of clarified lysates based on the absorption at 260 nm were layered onto 10%–50% sucrose gradient (prepared in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 50 μg/mL CHX) and centrifuged in an SW41-Ti rotor at 40,000 rpm for 2 h at 4°C. Fractions were collected using a Bio-Rad fraction collector, and the amount of total RNA in each fraction was measured using a NanoDrop spectrophotometer [66 (link)].

The crude polysaccharide was subjected to anion-exchange chromatography using a DEAE Sephacel resin (# 17-0500-01, Cytiva) column (2.5 × 20 cm, # Bio-Rad Laboratories) equilibrated with 50 mM sodium acetate. Fifty millimolar sodium acetate with 3 M NaCl was used as second mobile phase to generate a salt gradient. The gradient produced by a gradient mixer (# GM-1, Pharmacia Fine Chemicals) was applied over 24 h and ranged from 0 to 3 M NaCl in 50 mM sodium acetate. The flow rate was set to 3.5 ml/10 min/fraction collected in the fraction collector (# 2110, Bio-Rad Laboratories). Fractions of different anionic content were monitored by DMB (# Sigma-Aldrich) (Fig. S1A).

To evaluate the molecular weight distribution of PPH, the size exclusion chromatography (SEC) was performed according to Sae-Leaw et al. [27 ] using mixed standards with the molecular weight of 12.4 kDa (cytochrome c from horse heart), 29 kDa (carbonic anhydrase from bovine erythrocytes), 66 kDa (albumin, bovine serum), 150 kDa (alcohol dehydrogenase from yeast), and 200 kDa (β-amylase from sweet potato). Void volume was measured using blue dextran (2,000,000 Da). All samples (8.0 mg/mL) was diluted using a phosphate buffer saline (PBS, pH 7.4). Chromatographic separations of standards and samples (1,000 μL) were performed with a Sigma Andrich Superdex^® column (200 Increase 10/300 GL Cytiva, 28-9909-44, column L × I.D. 30 cm × 10 mm, 8.6 μm particle size). A low-pressure chromatography system (<1.3 mPa) and a fraction collector (Bio-Rad Laboratories, Hercules, CA) were used. For elution, PBS was used at a flow rate of 0.4 mL/min. Fractions (3 mL) were taken for absorbance measurement at 220 and 280 nm.