Imagej software

We seeded BMMs onto bone slice of sterile bovine into 96‐well plates at a density of 1 × 10⁴ cells/well. The cells were treated with different concentrations of sesamin (0, 5 and 10 μM), and then cells were induced every other day with complete α‐MEM supplemented with 30 ng/mL M‐CSF and 50 ng/mL RANKL. Once mature osteoclasts appeared in the control group, treatment of the cells was continued for 48 h. Osteoclasts attached to the bovine bone slices were subsequently removed using an ultrasonic oscillator. Then, bone resorption pits were observed by scanning electron microscopy (FEI Quanta 250, Hillsboro, USA) and the area of bone resorption was quantified using ImageJ software (Bethesda, MD, USA).

Protein was extracted from 786-O and Caki-1 cells after transfection for 48 h. Then, the samples were transferred to a polyvinylidene fluoride (PVDF) membrane, followed by successive incubation with 5% nonfat milk, primary antibodies and secondary antibodies. The relative expression of the target protein was analyzed with GAPDH as a loading control. The primary antibodies anti-TFE3 (ab196681, 1:1,000, Abcam, UK) and anti-GAPDH (60004-1-Ig, 1:5,000, ProteinTech Group, USA) were used in this research. After incubation with secondary antibody, the protein bands were imaged using ImageJ software (Thermo Fisher Scientific, Waltham, USA). The experiment was performed three separate times.

Nuclear extract from Jurkat cells was prepared using the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Scientific, Waltham, Massachusetts, USA). Electrophoretic mobility gel shift assays (EMSAs) were performed with LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Waltham, Massachusetts, USA) using 5 μg of nuclear extract and 0.6 ng biotin-labelled double-stranded oligonucleotides (50 bp fragment—Eurofins, Wolverhampton, UK). The sequences of the synthetic single-stranded oligonucleotides used in the construction of these double-stranded oligonucleotides are listed in the online supplementary methods.
Probes were prepared using a biotin 3′ end DNA labelling kit (Thermo Scientific, Waltham, Massachusetts, USA).
Single-stranded biotinylated oligonucleotides were mixed and annealed at room temperature for 1 h. Unlabelled competitor probes were used in 100-fold excess.
EMSAs were performed according to standard protocol (Thermo Scientific).
Band intensity was quantified with ImageJ software (Bethesda, Maryland, USA).
A detailed protocol is available online (see online supplementary methods section).

Transfected PTC cells were lysed in RIPA buffer (Solarbio, China), and phenylmethylsulfonyl chloride was used as a protease inhibitor to stabilize the whole lysate. The extracted proteins were quantified by the bicinchoninic acid assay (Thermo Scientific, United States). Then the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BioRad, United States) followed by transferring them to the polyvinylidene difluoride (PVDF) membranes (Millipore, United States). The primary antibodies were as follows: MVP (16478-1-AP, Proteintech), phospho-AKT^Ser473 (4060S, Cell Signaling Technology), total-AKT (4691S, Cell Signaling Technology), phospho-mTOR (381557, Zen Bioscience), total-mTOR (380411, Zen Bioscience), Phospho-p44/42 (4370T, Cell Signaling Technology), total-p44/42 (4695T, Cell Signaling Technology), phospho-p38 (4511T, Cell Signaling Technology), total-p38 (8690T, Cell Signaling Technology), and -Actin (AP0060, Bioworld Technology). Primary antibodies were used for immunoblotting at 1:1,000 dilution. The membranes were then incubated with a secondary antibody (ab97047 or ab6728, Abcam). Eventually, proteins were detected by the chemiluminescence kit (Thermo Scientific), and images of the protein bands were quantified by ImageJ software (NIH, United States).

The protein samples were extracted from VSMCs with RIPA lysis buffer (Beyotime) on ice for 30 minutes. After centrifugation at 12 400 g at 4°C for 10 minutes, the supernatants of VSMCs were harvested to determine the protein expression. The protein samples were mixed with the loading buffer and boiled at 95°C for 5 minutes. The boiled samples were separated on the SDS‐polyacrylamide gels, and the proteins were transferred to the polyvinylidenedifluoride membranes. These membranes were then incubated successively with 5% bovine serum albumin and the primary antibodies: Antibodies against GAPDH (1:1000 dilution), Runx2 (1:500 dilution), BMP2 (1:500 dilution), OCN (1:200 dilution), α‐SMA (1:1000 dilution), SM22α (1:1000 dilution), smoothelin (1:1000 dilution), LC3 (1:1000 dilution), P62 (1:1000 dilution) and Beclin‐1 (1:1000) overnight. The membranes were then washed with TBS‐T, followed by an incubation with a horseradish peroxidase‐conjugated secondary antibody (1:5000 dilution, CST) for 2 hours at room temperature. Protein blot bands were detected by ECL blotting substrate and then exposed to an X‐ray film. The densities of the bands were semi‐quantified and normalized against GAPDH by Image J software (Thermo Scientific).