Nuclear fractionation protocol

Cytoplasmic and Nuclear fractions were obtained using the nuclear fractionation protocol from Abcam. Cells were grown to 80–90% confluence and lysed into Buffer A (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, and 0.5% NP40) and then incubated on ice for 30 minutes. Lysates were spun down at 3000 rpm for 10 minutes at 4°C to pellet nuclei. The supernatant was removed as the cytoplasmic fraction and the nuclear pellet was washed 3 times in Buffer A to remove potential contaminants. The nuclear pellet was lysed in Buffer B (5 mM HEPES, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, supplemented with 300 mM NaCl added fresh). To ensure lysis, the nuclear pellet was passed through a 25-gauge needle and the lysates incubated on ice for 15 minutes. Lysates were then spun down at 16,000 g for 15 minutes to pellet insoluble debris. The supernatant was collected as the nuclear fraction. Fraction purity was assessed by western blotting with an antibody to Histone 2B.

For Sox11, cells were fractionated into nuclear and cytoplasmic pools using nuclear fractionation protocol from Abcam. Buffer A [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT and 0.05% NP40 (pH 7.9)] was used for the cytoplasmic fraction, whereas Buffer B [5 mM HEPES, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol (v/v) (pH 7.9) and 4.6 M NaCl] was used for the nuclear fraction. For other downstream targets, total lysate was collected using RIPA buffer (Upstate) containing 1% NP-40, protease-inhibitor cocktail (Calbiochem) and PhosSTOP (Sigma-Aldrich). The following dilutions of primary antibodies were used for the western blot analysis: 1:500 rabbit anti-Sox11 (Abcam, Ab134107); 1:500 Rab anti-fibronectin (Abcam, Ab2413); 1:500 Rab anti-N-cadherin (Cell Signaling Tachnology, D4R1H); 1:500 rat anti-E-cadherin (Abcam, Ab11512); 1:500 Rab-anti-Mex3A (Abcam, Ab79046); 1:500 Rab anti-Rcor2 (Abcam, Ab37113); 1:1000 Rab anti-GAPDH (Cell Signaling Technology, D16H11); and 1:1000 Rab anti-lamin B1 (Abcam, Ab16048). For detection, an enhanced chemiluminescence detection kit (Bio-Rad) was used.

Nucleus fractionations were performed according to the Nuclear Fractionation Protocol (Abcam, UK). Briefly, MSCs were harvested at indicated times after treatment with tBHQ (25 μM). The cells were mixed with 400 μL of lysis buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.05% NP40, pH 7.9, containing protease and phosphatase inhibitor cocktail), thoroughly scraped and incubated on ice for 10 min. The mixture was centrifuged for 10 min at 3,000 rpm, 4°C. The supernatant contains the cytoplasmic fraction. Next, the nuclear pellets were lysed with 120 μL of buffer (5 mM HEPES, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 26% glycerol, pH 7.9, containing protease and phosphatase inhibitor cocktail). 4 M NaCl was added to make its concentration 300 mM and the homogenized pellet was incubated on ice for 30 min at 14,000 × g, 4°C, and the resulting supernatant contains the nuclear fraction. Each fraction was quantified with western blotting.