Infinite m2000

Two hundred cotton plants were divided into 3 groups at each experimental site and 10 cotton plants were randomly selected from each group for marking for each cotton variety. Cotton was sampled at the bud, boll, and boll opening stages. A complete leaf from the top of each plant was collected. The leaf was immediately placed in a 10-ml sealed centrifuge tube after being detached and stored in a liquid nitrogen tank. After reaching the laboratory, the samples were stored in a deep-freezer at −70°C. The expression of Cry1Ab/c protein in Zhong30 cotton was quantified using a QualiPlate Kit for Cry1Ab/Cry1Ac (EnviroLogix Inc., Portland, ME, USA). About 20 mg of tissue sample was weighed and its exact mass was recorded. The test was done according to the manufacturer’s instructions. The optical density (OD) was measured at 450 nm using a microplate reader (Infinite M2000; Tecan Group Inc., Männedorf, Switzerland). The calibration was done by generating a standard curve of OD against the protein content using the following concentrations of the Bt protein (Cry1Ab) standard (EnviroLogix Inc.): 0.03125, 0.0625, 0.125, 0.25, 0.5 ng, 1.0, 2.0, and 4.0 ng mL⁻¹. The level of Cry1Ab/c protein in the fresh cotton leaf samples was determined using the standard curve and the dilution ratios of the extract (μg g⁻¹ FW).

Seeding of MC3T3-E1 cells (1.2 × 10⁴ cells/well) was in 6-well plates. In vitro and in vivo determination of ALP activity was performed applying an ALP staining kit (Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. Subsequently, measurement of OD values was at 540 nm applying a microplate reader (Infinite™ M2000; Tecan Group, Ltd.), and observation and collection of images were implemented under an inverted fluorescence microscope (Olympus Corporation).

SMSCs (1 × 10⁴ cells/well) were plated in a 6-well plate. ALP activity was determined using the ALP staining kit (Thermo Fisher Scientific, Inc.). Subsequently, optical density values were measured using a microplate reader (Infinite™ M2000; Tecan Group, Ltd.) at 540 nm, and images were collected under an inverted fluorescence microscope (Olympus).

Intracellular ROS levels were measured using the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Sigma-Aldrich). The cells were seeded in 96-well plates (#3596; Corning, Glendale, CA, USA), cultured overnight, and treated with CONA for 24 h. After this time, the cells were loaded with 10 μM of H2DCFDA and incubated at 37 °C for 30 min. Next, the fluorescence intensity was measured at a 488 nm excitation wavelength and 525 nm emission wavelength using a microplate reader (Infinite M2000, Tecan, Männedorf, Switzerland).

FAM-labeled SNORD50A/B and scrambled RNA were obtained from Gene Pharma (Shanghai, China). All fluorescence polarization assays were done using black, low-protein-binding 384-well plates in a total volume of 50 μL per well. Each well contains 5 μM SNORD50A or SNORD50B and gradient diluted proteins (TRIM21 alone, GMPS alone, and TRIM21 + GMPS) with a total concentration from 24 to 0.025 μM in Tris buffer. After a gentle mixing and incubation for 3 h, FP readings were taken at 470 nm (excitation) and 530 nm (emission) wavelengths on a Tecan Infinite M2000 fluorescence plate reader.