Microcal origin software

MTT assay was performed on a Synergy H1 multiplate reader (Biotek, USA) using H1299 cells containing engineered-inducible wild-type p53 gene, encoding p53 protein tagged with GFP protein. Overexpression of Mdm2 and MdmX was achieved by transfecting pCMV plasmids harboring a gene encoding the full-length Mdm2 and MdmX tagged with RFP protein. Cells were grown at 37 °C with 5% CO₂ in Dulbecco’s modified eagle medium supplemented with 10% serum, penicillin and streptomycin for 3 days, refreshed the medium with RPMI 1640 supplemented with 3% FBS. Cells were plated at a density of 1 × 10⁴/cm². After 2 h, added H203 and nutlin-3a for 24 h. After adding 10 µl/well MTT (5 mg/ml) solution, the cells were incubated for another 4 h at 37 °C in a CO₂ incubator. After the supernatant was discarded, the cells were washed with PBS, and 100 µl DMSO was added to each well. The plates were agitated on a plate shaker for 10 min and then were read the OD at 520 nm. Data were processed with MicroCal Origin software (v2017, MicroCal, USA).

Thyroid receptor α (A/B + DBD) and TRα (DBD), purified by SEC, were used for isothermal titration calorimetry (ITC) measurements using VP‐ITC MicroCal™ (MicroCal Inc., Northampton, MA, USA). Protein and ligand were prepared in 50 mm HEPES, pH 7.5, 125 mm NaCl, 5 mm MgCl₂ and 1 mm TCEP. For titration experiments, protein concentration ranged from 30 to 45 μm and ligand DR4 TRE: 5′‐CCAGGTCATTTCAGGTCAG‐3′ concentration ranged from 300 to 400 μm. Both protein and ligand were degassed for 5–10 min. The experiments were initiated by injecting 28 × 10 μL aliquots of DR4 TRE from the syringe into the calorimetric cell containing 1.5 mL of protein solution. All the titrations were performed at 25 °C and the buffer (pH adjusted to 7.5 at 25 °C). The change in thermal power as a function of each injection was automatically recorded using microcal origin software and the raw data were further processed to yield binding isotherms of heat released per injection as a function of molar ratio of DR4 TRE to TRα (A/B + DBD) or TRα (C domain). The data were acquired and processed using the microcal origin (MicroCal Inc.) software. Data were collected in triplicate.

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) software version 12 for Windows (SPSS Inc., Chicago, IL, USA). Student t-tests were used to determine the statistical significance of the differences between the experimental groups. A P-value of <0.05 was considered significant. Graphs were created using Microcal Origin software (version 3.78; Microcal Software, Inc., Northampton, MA, USA).

The ITC experiments were done using a VP-ITC microcalorimeter (MicroCal, LCC). All details for determination of binding constants for small effector molecules using ITC are fully described in the supplementary information. Heat isotherms of the dilution of the ligand in the cell buffer were collected in a blank run for each experiment in the absence of protein. The binding isotherms were calculated from received data and fitted to one-site and three-sequential binding sites models using the MicroCal ORIGIN software (Northampton) as described^{9 (link)}. All titrations were performed in duplicates with different purification batches of recombinant PII proteins. The association binding constant (K_a) was generated from the software by de-convolution and curve fitting. For calculation of dissociation constant (K_d), the K_a value was inversed.