Pgfip piston gradient fractionator model 152

The details of the polysome profiling analysis were described previously⁶³ . Briefly, 1 × 10⁷ cells were treated with 100 μg/mL cycloheximide in DMSO for 5 min at 37 °C and then harvested for polysome profiling. The cells were lysed in 500 μl of polysome lysis buffer and then centrifuged. The supernatant was then loaded onto a 5–50% (w/v) sucrose density gradient, ultracentrifuged at 20,000 × g for 2 h at 4 °C in a Beckman SW41 rotor and subsequently fractionated using a BioComp PGFip Piston Gradient Fractionator Model 152. The absorbance at 254 nm was measured using an absorbance detector connected to the fraction collector. RNA was extracted from different fractions, and RT-qPCR was conducted to evaluate the distribution of targets.

293T cells were plated in 15-cm plates and transfected with circ-SMO overexpression plasmid or circ-SMO noATG plasmid. After 48 h, the cells were treated with 100 μg/mL cycloheximide in DMSO for 5 min at 37 °C, washed twice with ice-cold 1× PBS containing 100 μg/ml cycloheximide and then harvested by trypsinization for polysome profiling. Cells were lysed in 500 μl polysome lysis buffer (5 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 1.5 mM KCl, 1× protease inhibitor cocktail (EDTA-free), 0.5% Triton X-100, 2 mM DTT, 0.5% sodium deoxycholate, 100 units RNase inhibitor, and 100 μg/ml cycloheximide] on ice for 15 min, followed by centrifugation at 4 °C for 7 min at 16000×g to pellet nuclei and mitochondria. The supernatant was then loaded onto a 5–50%(w/v) sucrose density gradient and ultracentrifuged at 20,000×g for 2 h at 4 °C in a Beckman SW41 rotor and subsequently fractionated using BioComp PGFip Piston Gradient Fractionator Model 152. Absorbance at 254 nm was measured using an absorbance detector connected to the fraction collector. RNA was extracted from fractions using TriZol LS solution, and RT-qPCR was conducted to evaluate the Circ-SMO and SMO mRNA levels in indicated fractions.

293T cells were first transfected with the circEZH2 OV plasmid. Forty-eight hours later, the cells were treated with 100 μg/ml cycloheximide (CHX) in DMSO for 5 min at 37 °C, washed with CHX-containing PBS and harvested for subsequent polysome profiling. Cells were lysed in 500 μl of polysome lysis buffer [5 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 1.5 mM KCl, 1× EDTA-free protease inhibitor cocktail, 0.5% Triton X-100, 2 mM dithiothreitol (DTT), 0.5% sodium deoxycholate, 100 units RNase inhibitor and 100 μg/ml CHX] on ice for 15 min and were then centrifuged at 4 °C and 16,000 × g for 10 min. The supernatant was then collected and overlaid onto a 5–50% (w/v) sucrose density gradient, ultracentrifuged at 4 °C and 20,000 × g for 2 h in a Beckman SW41 rotor and subsequently fractionated using a BioComp PGFip Piston Gradient Fractionator Model 152. The absorbance at 254 nm was measured using an absorbance detector connected to the fraction collector. RNA was extracted from fractions using TRIzol LS solution, and RT-qPCR was conducted to evaluate the circEZH2 and EZH2 mRNA levels in the indicated fractions.