Annexin 5 fitc solution

The Populus protoplasts from SDX isolation were carried out as previously described by Lin et al. (2013 (link))and Wang et al. (2020 (link)) with minor modifications. Briefly, the debarked stem segments of 3-month-old WT and PagRabE1b transgenic poplars were incubated in a cell wall digestion enzyme solution [1.5% (wt/vol) Cellulase R-10 and 0.4% (wt/vol) pectolyase Y-23 in 20-mM MES,0.6-M mannitol and 20-mM KCl solution, 10-mM CaCl₂, and 0.1% (wt/vol) BSA, pH 5.7] for 40 min in the dark at room temperature. Protoplasts were filtered through a 70-μm cell strainer and spun down at 150 × g for 5 min. Protoplasts were resuspended in W5 solution (2-mM MES, pH 5.7, 125-mM CaCl₂, 154-mM NaCl, 0.1-M glucose, and 5-mM KCl). The isolated protoplasts were resuspended in a 195-μl prediluted binding buffer, and 5-μl Annexin V-FITC solution (Solarbio, Beijing, China) was added, mixed, and incubated for 30 min at room temperature, and then added 1 μl of the 20-μg ml⁻¹ propidium iodide (PI) storage solution. After the addition of another 300-μl binding buffer, the suspended protoplasts were analyzed using a BD Aria SORP cell sorter (BD Biosciences, USA) with 488-nm excitation for FITC and 530 nm for PI. Three independent sets of experiments were performed.

Poly-l-lysine medium was purchased from Sigma–Aldrich, St. Louis, MO, U.S.A.; preadipocyte medium (PAM, C-27410), preadipocyte differentiation medium (PADM, C-27436) and adipocyte medium (ADM, C-27438) were obtained from PromoCell, Heidelberg, Germany; Oil Red O kit, annexin V-FITC solution and propidium iodide solution were bought from Solarbio, Beijing, China; Cell Counting Kit-8 (CCK8) was purchased from KeyGen Biotech, Nanjing, China; Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL) assay was obtained from Vazyme Biotech, Beijing, China. All the antibodies used were purchased from Abcam Co., Ltd., Cambridge, U.S.A.; TRIzol reagent and TaqMan MicroRNA reverse transcription kit were bought from Invitrogen, Waltham, MA, U.S.A. All the primers and siRNAs were synthesized at GenePharma, Shanghai, China.