Chemicals and Antibodies
Recombinant murine TNF-α was purchased from PeproTech(Rocky Hill, NJ, USA). Actinomycin D was obtained from MedChem Express (Princeton, MA, USA). Antibodies for phospho-ERK1/2 (p-ERK1/2, Thr202/Tyr204), total ERK1/2, phospho-JNK (p-JNK, Thr183/Tyr185), total JNK, phospho-p38 MAP kinase (p-p38, Thr180/Tyr182), total p38 MAPK, phospho-nuclear factor-kappa B p65 (p-p65, Ser536) and total p65 were purchased from Cell Signaling Technology (Danvers, MA, USA).
Antibodies for Fto were purchased from Abcam (Cambridge, MA, USA) and Proteintech (Wuhan, Hubei, China).
Apical periodontitis induction
All procedures were in accordance with the Ethics Committee of School and Hospital of Stomatology, Wuhan University, China. As in previous studies[32, 33], 8-week-old mice(C57BL/6J) were used for induction of apical periodontitis. In brief, animals were fixed on the table after being anesthetized. The right mandibular first molar pulp was opened using a controlled rotation handpiece with a No.1/4 round bur. Control groups were no surgical interventions on the left mandibular first molar. Animals were euthanized with excessive CO2 for following experiments after the pulp champers were exposed for 14 days.
Histological and immunohistochemical analysis
The mandibles and the periapical tissues were carefully extracted and fixed with 4% paraformaldehyde for 24 hours at room temperature. After decalcification with 10% ethylenediaminetetraacetic acid solution for 1.5 months, tissues were dehydrated, embedded in paraffin, and sectioned at 5μm thickness. Hematoxylin and eosin (HE, Servicebio, China) were performed according to the manufacturer’s instructions. Immunohistochemical analyses for Fto (1:100, Servicebio, China) were also performed. The expression of Fto in cementoblasts and ambient periodontal tissues were verified and photographed with a microscope.
Cell culture and treatment
An immortalized cementoblast cell line OCCM-30 was generously provided by Dr. Martha J. Somerman (National Institutes of Health, MD, USA), which was cultured in Dulbecco's modified Eagle's medium (DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum(FBS, Gibco) in 5% CO2 at 37°C as previously described. The medium was changed to DMEM with 5% FBS when TNF-α (Peprotech) or actinomycin D (MCE) were applied. Actinomycin D was dissolved in dimethyl sulfoxide (DMSO) and the final concentration was less than 0.1%. HEK293T was cultured in DMEM (Hyclone), supplemented with 10% FBS(Gibco).
Plasmid construction, lentivirus production, and cell infection
pLKO.1-puro vector was used to knockdown Fto, and the shRNA against Fto and negative control(NC) was provided by MiaoLingBio. The shFto and shNC sequences were listed in Table 1. Lentiviruses were generated in HEK293T cells which were transfected by Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific) with viral packaging constructs pMD2.G (Addgene) and pSPAX2 (Addgene). The lentiviral supernatant was collected every 24 hours and filtered through a 0.45 μm membrane (Millipore). The lentiviruses were used to infect OCCM-30 cells for 48 hours, then the cultured medium was changed by selective medium supplemented with blasticidin (20 µg/mL) or puromycin (6 µg/mL) in accordance with the plasmid instructions. The survived cells were used for in vitro experiments. Infection efficiency was measured by real-time quantitative polymerase chain reaction (qRT-PCR) and western blot.
Quantitative Real-time Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted by FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme), and cDNA was synthesized with a HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme). qRT-PCR was conducted on a PCR system (Bio-Rad, Hercules, CA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme). The 2-ΔΔCT method was used to normalize the expression of target genes to Gapdh or β-Actin. Primer sequences were shown in Table 2.
The protein samples were harvested using M-PER mammalian protein extraction reagent(Thermo Fisher Scientific) supplemented with protease inhibitors(Roche) and phosphatase inhibitors (Roche), and centrifugated with high speed at 4 °C. The supernatant was collected to examine the protein concentration using a BCA assay kit (Biosharp) and stored at -20 ℃. Cell lysates (20-30μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). After being blocked with 5% nonfat milk, the membrane was incubated with primary antibodies against Fto (Abcam or Proteintech), phospho-ERK1/2 (Thr202/Tyr204, CST), total ERK1/2, phospho-JNK (Thr183/Tyr185, CST), total JNK, phospho-p38 MAP kinase (Thr180/Tyr182, CST), total p38 MAPK, phospho-nuclear factor-kappa B p65 (Ser536, CST) and total p65 overnight at 4°C. β-Actin (Servicebio) or GAPDH (Servicebio) was used as the loading control. The membrane was incubated with the WesternBright® ECL kit (Advansta) and scanned by the Odyssey LI-COR scanner. ImageJ software was used to analyze the images.
Enzyme‐linked immune sorbent assay (ELISA)
OCCM-30 cells were stimulated with 20 ng/mL TNF-α for 12h, and the supernatants were collected, centrifugated, and stored at -80°C. The concentrations of mouse Il-6 were measured using an ELISA kit (Dakewe Bio-engineering, China) following the product instructions.
mRNA Stability Assay
OCCM-30 cells were treated with 20 ng/mL TNF-α for 6h following stimulated with 10µg/mL actinomycin D (MCE) for 0, 2, and 4 h. Total RNA was reverse-transcribed into cDNA, and qRT-PCR was conducted to measure the mRNA stability. The mRNA transcript levels were normalized to 0h.
A two-tailed unpaired Student’s t-test was applied to statistical analysis. P values of less than 0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 7.0. In the figures, values were presented as mean ± SEM, ∗P < 0.05, and ∗∗P < 0.01.