Isolation of tenocyte cytoplasmic and nuclear extracts

Isolation of cytoplasmic and nuclear extracts was performed as previously described.⁹ Briefly, tenocytes were trypsinized and washed twice in 1 ml of ice-cold PBS. The cell pellets were re-suspended in 400 μl lysis buffer containing protease inhibitors and 10% Nonidet P-40 (ST2045, Beyotime Biotech Inc., Shanghai, China), and incubated on ice for 15 min. The cell suspension was mixed vigorously for 15 s then centrifuged for 1.5 min at 14,000 × g. The supernatants were harvested as the cytoplasmic extracts. The pellets were then re-suspended in 25 μl ice-cold nuclear extraction buffer (P0027, Beyotime Biotech Inc, Shanghai, China), incubated for 30 min with intermittent mixing, then centrifuged. The supernatant were harvested as described for the nuclear extracts.