Fig 1: LepR-Cre mediates recombination in NP cells of IVD tissues at postnatal stages. (A) Diagram of the timeline analysing the specificity of LepR-Cre in NP cells of the IVD of LepR-Cre; R26R; TdTomato mice.(B) Quantification of the percentage of Tdtomato+ cells to all NP cells from time points P0, P3, P10, P1M, P2M, P3M, and P6M. (C) Quantification of the percentage of Tdtomato+ cells to all LepR+ cells in the aforementioned time points. (D) Representative immunofluorescence staining images of the L4–L5 IVD using antibodies against LepR. P = postnatal. Scale bars, 100 ?µm (n ?= ?5 mice per group). Data are presented as mean ?± ?SD. * indicates p ?< ?0.05, and NS indicates not significant, as determined by ANOVA with Bonferroni post hoc analysis. ANOVA = analysis of variance; IVD = intervertebral disc; LepR = leptin receptor; NP = nucleus pulposus; SD = standard deviation.
Fig 2: LepR-expressing NP cells are closely associated with the turnover of matrix proteins in the IVD. (A) Representative immunofluorescence staining images of the L4–L5 IVD from P3M LepR-Cre; R26R; Tdtomato mice using antibodies against MMP13. (B) Representative immunofluorescence staining images of the L4–L5 IVD from P3M LepR; R26R; Tdtomato mice using antibodies against aggrecan. Scale bars, 100 ?µm. Images on the right are magnification of certain areas in the merged images. Scale bars, 20 ?µm. Arrowheads indicate matrix protein closely associated with LepR-expressing NP cells; arrows indicate matrix protein distant to LepR-expressing NP cells (A and B) ?(n ?= ?5 mice per genotype from 3 independent experiments). IVD = intervertebral disc; LepR = leptin receptor; NP = nucleus pulposus.
Fig 3: DNA methylation in the promoter region of the LEPR gene. a A schematic representation of the LEPR gene, including the CpG islands in the promoter region. b The methylation pattern in the LEPR promoter on the fetal and maternal sides of third-trimester placental biopsies from the control group. c The % methylation level in the LEPR promoter region from third-trimester placenta. DNA was extracted from third-trimester placental biopsies (on the fetal and maternal sides) in the control and obese women. After bisulfite treatment, the methylation level was determined by pyrosequencing. The data are quoted as the mean ± SEM. Statistical significance was assessed in a Friedman test
Fig 4: LEP and LEPR expression levels in human third-trimester placental tissue. a, b mRNA expression of LEP and LEPR. Total RNA was extracted from the fetal and maternal sides of third-trimester placenta in control and obese women, and then analyzed using RT-qPCR. The data are quoted as the mean ± SEM. *p < 0.05 in a Wilcoxon test. (a) Maternal side vs. fetal side. c Leptin protein expression. Third-trimester placental biopsies were fixed, embedded in paraffin, and stained for anti-leptin antibody, as described in the “Methods” section. The data are quoted as the mean ± SEM of N = 16 placental sections. *p < 0.05; **p < 0.01 in a Wilcoxon test. (a) Maternal side vs. fetal side. d LEPR protein expression. Lysates of third-trimester placental biopsies from control and obese women were extracted and then subjected to Western blot analysis using an anti-LEPR or anti-ß-actin antibody, as described in the “Methods” section. The data are representative of five separate experiments and are quoted as the mean ± SEM. **p < 0.01 in a Student t test. (b) The obese group vs. the control group
Fig 5: The percentage of human LepR+ NP cells significantly decreases during disc degeneration. (A) Representative immunofluorescence staining images of NP cells from human IVD samples of Grades II to V using antibodies against LepR. (B) Quantification of the percentage of LepR+ cells to all notochord-derived cells in different time points. Scale bars, 20 ?µm (n ?= ?3 per group). Data are presented as mean ?± ?SD. * indicates p ?< ?0.05, and NS indicates not significant, as determined by ANOVA with Bonferroni post hoc analysis. ANOVA = analysis of variance; DAPI = 4,6-diamino-2-phenylindole; IVD = intervertebral disc; LepR = leptin receptor; NP = nucleus pulposus; SD = standard deviation.
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