Dexamethasone (dex)

For adipogenic differentiation, porcine MSCs (1 × 10⁵/well) at P4 were seeded in 6-well plates until they reached 70%–80% confluence. These cells were first cultured in adipogenic induction medium for 3 days and sequentially in maintenance medium for another 3 days. Next, the two media were replaced alternately until 21 days. Adipogenic induction medium is composed of DMEM/F-12 supplemented with 10% FBS, 10 μM of dexamethasone (Solarbio, Beijing, China), 200 μM of indomethacin (Solarbio, Beijing, China), and 10 μM of insulin (Solarbio, Beijing, China), while maintenance medium is composed of basal medium supplemented with 0.2 nM of insulin. After 21 days, the cells were fixed with 4% paraformaldehyde and then stained with oil red O (Solarbio, Beijing, China).
For osteogenic differentiation, porcine MSCs (1 × 10⁵/well) at P4 were seeded in 6-well plates until they reached 80–90% confluence. The medium was replaced with osteogenic induction medium. Osteogenic induction medium is composed of basal medium supplemented with 0.1 μM of dexamethasone (Solarbio, Beijing, China), 10 μM of β-glycerophosphate (Coolaber, Beijing, China), and 50 μM of vitamin C (Solarbio, Beijing, China). The media were changed every 2–3 days. After 21 days, the cells were fixed with 4% paraformaldehyde and then stained with alizarin red S (Solarbio, Beijing, China).

Cells were kept in a growth medium (GM) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), which was stored in an incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂ environment. We used a differentiation medium (DM) containing Dulbecco’s modified Eagle’s medium (DMEM; VivaCell, Shanghai, China) supplemented with 10% FBS, 1 μmol/L dexamethasone (DEX; Solarbio, Beijing, China), 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX; Solarbio, Beijing, China), and 10 μg/mL insulin (Solarbio, Beijing, China). The maintenance medium (MM) consisted of DMEM with 10% FBS and 10 μg/mL insulin. According to the manufacturer’s instructions, transfection was carried out using the lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA). The transfection reagent was mixed with RNA oligo (let-7a-5p mimic, inhibitor, negative control (NC), inhibitor negative control (INC)) and siRNA (si-Srebf2, si-Thbs1, and si-NC), and then the mixture was transfected into the cells. The concentration of transfection was 50 uM for the mimic and NC, and 100 uM for the inhibitor, INC, siRNA, and si-NC. The above RNA oligo and siRNA were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and Tsingke Biotechnology Co., Ltd. (Beijing, China), respectively, and Table S5 displays the sequence information.

For the osteogenic differentiation assay, PDLSCs were cultured in osteogenic medium containing α-MEM (BI), 10% FBS (BI), 10 nM dexamethasone (Solarbio), 10 mM β-glycerophosphate (Solarbio) and 50 mg/L ascorbic acid (Solarbio). After 4 weeks, the cells were fixed with paraformaldehyde and stained with Alizarin Red solution (Sigma, St. Louis, MO, USA) to detect mineralized nodules. To analyse the intensity of Alizarin red staining, mineralized nodules were dissolved in 10% cetylpyridinium chloride (Solarbio) and quantified using a microplate reader at 562 nm. When necessary, Ly294002 (#9901, CST) (an inhibitor of Akt phosphorylation) or SC79 (HY-18749, MCE, USA) (a promoter of Akt phosphorylation) was added to the osteogenic medium at concentration of 10 µmol/L or 5 µg/mL, respectively.
For the adipogenic differentiation assay, PDLSCs were cultured in adipogenic medium containing α-MEM (BI), 10% FBS (BI), 1 µM dexamethasone (Solarbio), 0.2 mM indomethacin (Solarbio), 0.01 g/L insulin (Solarbio) and 0.5 mM isobutyl-methylxanthine (Solarbio). Four weeks later, the cells were fixed with paraformaldehyde and stained with oil red O (Solarbio) to detect lipid droplets.

Osteogenesis and lipogenesis induction were used to detect the multidirectional differentiation ability of PDLSCs. The osteogenic induction solution formulation consisted of D-MEM (Hyclone, USA), 10% FBS, 10 mmol/L sodium β-glycerate (Macklin, Shanghai, China), 0.1 μmol/L dexamethasone (Solarbio, Beijing, China), and 50 mg/L vitamin C (Solarbio, Beijing, China). Osteogenic induction solution was changed every 3 days and maintained for 28 days. Then, cells were fixed by paraformaldehyde and stained by alizarin red solution (Solarbio, Beijing, China). The lipogenic induction solution was formulated with D-MEM, 10% FBS, 10 μmol/L dexamethasone (Solarbio, Beijing, China), 200 μmol/L indomethacin (Solarbio, Beijing, China), 0.5 mmol/L 3-isobutyl-1-methylxanthine (IBMX) (Sigma, USA), and 10 mg/L insulin (Solarbio, Beijing, China). Cultured for 28 days, the cells were fixed and stained by oil red O solution (Solarbio, Beijing, China). Calcium nodules and lipid droplets were observed under a fluorescent inverted microscope (Olympus, Japan).