Cell culture

The cementoblast cell line (OCCM-30) originated from murine cementoblasts and was provided by Dr. Martha J. Somerman. Cells were kept in DMEM medium (Hyclone, USA) with 10% FBS (Every Green, China). For mineralization induction, cells were cultured in OIM with 5% FBS, ascorbic acid (50 μg/mL; Sigma-Aldrich, USA), and Naβ-glycerophosphate (10 nmol/L; Sigma-Aldrich, USA) for 0, 4, and 7 days.

RNA sequencing and data analysis

After culturing the cells in plates, TRIzol (Takara, Japan) was added to extract RNA. The Beijing Genomics Institute (BGI; China) conducted the sequencing and analysis of the RNA production. Differentially expressed genes (DEGs) were screened by the criteria |log2(FC)| > 1 and FDR < 0.01. The pheatmap package was used to generate heat maps. Differential genes were annotated through NCBI, Uniprot, Gene Ontology (GO), and other databases, with GO enrichment conducted using the ClusterProfiler R package.60

qRT-PCR

Use PrimeScript™ RT Master Mix (Takara, Japan) reagent to reverse transcribe RNA into cDNA. qRT-PCR was conducted in triplicate on an Applied Biosystems QuantStudio 6 using SYBR qRT-PCR Master Mix (Vazyme, China). The protocol process included: initial denaturation (95 °C, 30 s), followed by 40 cycles (95 °C, 10 s) and 62 °C for 34 s. Primers were provided by Sangon Biotech (China, see Table S1). Gene expression fold changes were normalized to β-actin using the 2–ΔΔCT method.

Western blotting

Proteins from cells were obtained using M-PER lysis buffer (Merck, Germany) and quantified with BCA kit (Beyotime, China). Samples were mixed with SDS loading buffer (Epizyme, China). Protein samples of 20–40 µg were applied onto SDS-PAGE gels (ACE, China) for electrophoresis. After electrophoresis, samples were transferred onto PVDF membranes (Millipore, USA), after which 5% skim milk was applied for blocking. Incubated with primary antibodies (refer to Table S2) overnight at 4 °C and secondary antibodies for 1 h at room temperature, samples were visualized with super-enhanced chemiluminescence substrate (Biopmk, China) and imaged on an Odyssey LI-COR scanner (USA).

Animal models

The study used male C57BL/6 mice of varying ages (3 weeks, 6 weeks, and 6 months; weights: 9–32 g; n = 8 per group), with all protocols approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval No. S07921060E). Following euthanasia by CO₂ asphyxiation, mandibles were collected.

3-week-old male C57BL/6 mice (9–12 g) were randomly divided into a healthy control and a GSK-J4 group (12 mice per group). GSK-J4 (10 mg/kg) was administered intraperitoneally three times a week for 5 weeks. Mice were euthanized at 8 weeks, and mandibles were collected. All procedures were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (Approval No. S07923050C).

Male BALB/c-nu nude mice, 6 weeks old and weighing 19–20 g, were used in the study. The study was approved by the Animal Experimentation Ethics Committee of Wuhan University (Approval No. WP20230603). Gelatin sponges with sh-NC, sh-Kdm6b, or sh-Kdm6b + lactate sodium OCCM-30 cells were implanted subcutaneously, with four implantation sites per mouse. From day 3 post-surgery, the sh-NC and sh-Kdm6b groups received subcutaneous PBS injections three times weekly, while the sh-Kdm6b + lactate sodium group received sodium lactate (2 g/kg, 50 μL) every other day. After 4 weeks, cell-tissue composites were collected post-euthanasia. All animal experiments complied with the updated ARRIVE guidelines for preclinical animal studies.

Immunohistochemistry and immunofluorescence staining

For IHC, sections were prepared using the UltraSensitive SP IHC Kit (MXB Biotech, China). Samples were treated over 12 h at 4 °C with specific primary antibodies (see Table S2). Cementoblast morphology was observed and imaged microscopically. For IF assay, samples were pre-treated with goat serum at 37 °C for 1 h. Incubate overnight with different primary antibodies (see Table S2) at 4 °C. They were subsequently treated with FITC-conjugated secondary antibody (ABclonal, China) and 594-conjugated Goat anti-Rabbit IgG (ABclonal, China) for 1 h. The working diluted ratio of secondary antibody was at 1:200. Nuclei were counterstained with DAPI dye (ZSGB-BIO, China). Image capture was conducted with fluorescence microscopy.

ALP staining, ALP activity assay, and ARS staining

ALP staining was performed using ALP Assay Kit (Beyotime, China) on days 4 and 7 with OIM induction, followed by imaging. ALP activity was assessed usinga detection kit (Nanjing Jianchen, China). Fixed cells were dyed with 1% Alizarin Red Solution (OriCell, China) to visualize mineralized nodules, which were then photographed.

Transfections with siRNA, lentivirus, and plasmid

OCCM-30 cells underwent transient siRNA transfection with si-NC (GenePharma, China) and si-Pdk1 (5’- GCUGAGUAUUUCUUUCAAGUUTT-3’; GenePharma, China) using Lipofectamine 2000 (Thermo Scientific, USA), followed by a medium change after 6 h.

Sh-NC and sh-Kdm6b lentiviruses (GenePharma, China) were introduced at multiplicity of infection of 100 with Polybrene (5 ng/mL). After 24 h, original transfection medium was substituted with puromycin-containing medium (4 μg/mL; Beyotime, China) for 2–4 days to select cells.

To overexpress target genes, NC-, Pdk1-, and Zeb2-over plasmids (Miaoling Biotech, China) were transfected using TurboFect (Thermo Scientific, USA). A lysine-to-arginine mutation was introduced into the Zeb2 plasmid using the Mutation kit (Vazyme, China). Samples were collected post-transfection for further analysis.

ATP, glucose, and lactate measurements

For ATP measurement, cementoblasts were plated in a black 96-well plate at a density of 2 × 104 cells per well. After cultivating for 4 h, ATP content was then assessed using the CellTiter-Glo Luminescent Assay (Promega, USA) on a Tecan Spark microplate reader (Switzerland), with results normalized to cell count. For glucose consumption and lactate production assays, adherent cells were washed with PBS and incubated with phenol red-free DMEM (Gibco™, USA) for 24 h. The measurements of glucose levels were performed by the Glucose (HK) Assay Kit (Sigma-Aldrich, USA). Lactate production levels were assessed with an assay kit (Nanjing Jiancheng, China).

Seahorse assays

After seeding cementoblasts at a density of 5 × 104 cells per well in XF24 plates (Agilent, USA), ECAR and OCR were measured using the Agilent Seahorse XF Glycolytic Rate Assay Kit (Agilent, USA) and the XF Cell Mito Stress Test Kit (Agilent, USA).

For the Glycolytic Rate Assay, the working concentrations were Rotenone/Antimycin A (Rot/AA; 0.5 μmol/L) and 2-DG (inhibitor of glycolysis; 50 mmol/L). For the Cell Mito Stress Test, concentrations used 1.5 μmol/L oligomycin (ATP synthase inhibitor), 1 μmol/L FCCP (mitochondrial membrane potential uncoupler), and 0.5 μmol/L Rot/AA.

Double fluorochrome labeling

One week before euthanasia, mice received an intraperitoneal injection of calcineurin (5 μg/g; Merck, Germany), followed by alizarin complexone (20 μg/g; Merck, Germany) 48 h prior. Mandibular specimens were collected, fixed, and dehydrated in graded ethanol (70%–100%). Then samples were embedded in methyl methacrylate (Sinopharm, China) without decalcification. Samples were sectioned (10 μm) by Servicebio (China) and scanned with a whole-slide scanner. The distance between calcineurin and alizarin fluorescent bands was measured and analyzed using ImageJ.

Micro-CT scanning

Mouse mandibles were dissected, trimmed of excess muscle and incisors, and placed in PBS. The Bruker Skyscan 1276 Micro-CT system (Belgium) was preheated for 15 min, after which the samples were positioned on the holder. Pre-scanning and flat-field correction were performed. The mandibles were scanned using a voltage of 70 kV, a current of 114 µA, and a voxel resolution of 10 µm. A region of interest (ROI) was defined for each mandible, extending from 220 µm mesial to the mesial root of the first mandibular molar to 220 µm distal to the distal root. Image reconstruction was carried out using NRecon, with angle adjustment and ROI selection performed in DATA Viewer. Sagittal and coronal images of the ROI were then exported.

H&E, Masson, and Alizarin Red Staining

Specimens were fixed in paraformaldehyde for 1 day. Samples were then rinsed, decalcified, dehydrated, embedded, and sectioned. Sections were sent to Servicebio (China) for H&E staining, Masson trichrome and Alizarin Red staining. After staining, sections were dehydrated, mounted, and scanned using a digital pathology whole-slide scanner.

ChIP-sequencing and ChIP-qPCR

Cell samples were collected, cross-linked, lysed, and digested. Immunoprecipitation was conducted with an H3K27me3 antibody (Abcam, UK), followed by elution and DNA recovery per the Pierce™ Magnetic ChIP Kit (Thermo Fisher, USA) protocol. A portion of the product was sent to the BGI for analysis, with the remainder used for ChIP-qPCR validation (primer details in Table S3).

Molecular docking and sequence alignment

The homology model for ZEB2 was built using SWISS-MODEL. The L-lactate molecular structure was obtained from PubChem, and docking was conducted using CB-DOCK261 after preprocessing both molecules with MOE software to determine the small molecule-protein binding site.

ZEB2 protein sequences from different species were obtained from NCBI. Multiple sequence alignment was conducted using CLUSTALW, and the alignment results were presented using ESPript.

Lactylation proteomics

Cells were collected after 0 and 7 days of mineralization induction. After being washed thrice, the collections were centrifuged for 10 min (3 000 r/min; 4 °C). Cell pellets were snap-frozen in liquid nitrogen for 5 min after removing the supernatant. Samples were then sent to PTO BIO (China) for lactylation proteomics analysis.

Co-immunoprecipitation

Co-IP assays were conducted according to the instructions (Absin, China), using a Tag-FLAG antibody (1:50; CST, USA) for enrichment. The enriched products were then analyzed by Western blotting.

Statistical analysis

Each experiment was performed in triplicate. Data was conducted with GraphPad Prism 9 (GraphPad Software, USA) and shown as mean ± standard deviation. Statistical differences were assessed with t-tests and one-way ANOVA. The statistical significance is indicated by “*” for P < 0.05, “**” for P < 0.01, “***” for P < 0.001, and “****” for P < 0.000 1.