C3206

Alkaline phosphatase (ALP) staining assays were used to assess the effect of inhibition of cellular ferroptosis under high-lipid conditions on osteogenic differentiation of Mc3t3-E1cells. Mc3t3-E1 cell suspensions were added to 24-well plates (2 × 10⁴ cells per well) and incubated in a conventional incubator for 12 hours. The medium was then changed to regular medium, high-fat medium, and high-fat plus Ferrostatin-1 medium. After 24 hours of incubation, all media were changed to osteogenic differentiation induction medium, which was prepared with 50 μg/ml ascorbic acid (Sigma, A4544-25G), 10 nmol/l dexamethasone (MCE, HY-14648), and 10 mmol/l β-glycerophosphate (Sigma, G9422-10G). Seven days after osteogenic induction, half of the cells were stained for ALP using the ALP staining kit for the plates (Beyotime, C3206), and staining was performed according to the kit guidelines. Observation was made with a bright-field microscope. Photographs were then taken with a bright-field microscope ((OLYMPUS, BX63). The other half was assayed for ALP activity using the ALP quantification kit. The supernatant was collected, and the absorbance was measured at 405 nm (Beyotime, P0321S), and the cellular ALP activity was counted quantitatively.

In the osteogenic differentiation experiment, PDGFRβ;Td+ cells were induced for 7 days for ALP staining (C3206, Beyotime) and ALP activity evaluation (A059‐2‐2, Jian Cheng Technology, China), 14 days for Western blot assays, and 21 days for Alizarin Red staining (ALIR‐10001, Cyagen) unless otherwise specified. Assays were performed according to the manufacturer's instructions. Semiquantitative analysis of matrix mineralization was detected using cetyl‐pyridinium chloride (CPC; C9002, Sigma–Aldrich), and absorbance at 562 nm was measured with a microplate reader.
In the adipogenic differentiation experiment, PDGFRβ;Td+ cells were induced for 14 days for Western blot assays and Oil red O staining (OLIR‐10001, Cyagen). Assays were performed according to the manufacturer's instructions.

Mesenchymal stem cells were seeded in six‐well plates at a density of 5 × 10⁵ cells per well. When cells reached 100% confluence, the basal medium was changed into osteogenic induction medium: α‐MEM containing 20% FBS, 1% penicillin/streptomycin, 5 mM β‐glycerophosphate, 50 μg/mL ascorbic acid and 10 nM dexamethasone (all from Sigma‐Aldrich). The medium was refreshed every other day. For alkaline phosphatase (ALP) staining, after 10 days the medium was discarded, and the samples were washed with PBS twice and fixed with 4% paraformaldehyde (Sigma‐Aldrich). ALP staining was performed with a commercial kit (Beyotime, C3206) according to the manufacturer's protocol. Cells were cultured for 14 or 28 days, and the Alizarin red (Sigma‐Aldrich, A5533) staining was performed according to the manufacturer's instructions. Photographs were taken by an inverted optical microscope (Leica, M205FA). The 10% cetylpyridinium chloride was added for quantitative analysis, and the absorbance values were measured at 562 nm.

For alkaline phosphatase (ALP) staining, cells were cultured for 7 days in OΜ medium, fixed in 4% paraformaldehyde and stained with an ALP staining kit according to the manufacturer’s protocol (C3206, Beyotime, China). The formation of mineralized nodules of cells cultured for 21 days in OΜ medium were evaluated by Alizarin Red S staining (ALIR-10001, Cyagen, Taicang, China) and the lipid droplets of cells cultured in AΜ medium for 19 days were stained with Oil-Red O (OILR-10001, Cyagen, China).

The process of alkaline phosphatase (AP) staining for pluripotent stem cells was determined according to the manufacturer's instructions (C3206; Beyotime, Beijing, China).

Testes were embedded in O.C.T compound (Sakura Finetek, Torrance, CA), frozen in liquid nitrogen, and cut into 8-μm-thick sections. The probes (Table S1) were added to the sections and hybridized overnight at 60 °C. After washing and blocking at room temperature, sections were incubated with alkaline phosphatase (AP) conjugated anti-DIG Fab fragments overnight. Sections were cleaned with maleic acid buffer containing Tween 20 (MABT) solution and AP buffer. After adding chromogenic solution (Beyotime Biotechnology, C3206), the sections were washed with double-distilled water, dehydrated in gradient ethanol, cleared with xylene, mounted with SlowFade Gold antifade reagent (Life Technologies), and finally analyzed under a microscope (LEICA DM2500, Germany).