Journal: Cell reports

Article Title: Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling

doi: 10.1016/j.celrep.2020.108007

Figure Lengend Snippet: (A) Heatmap hierarchical clustering showing the gene expression profiles of Runx2 fl / fl (control) and Gli1 - Cre ERT2 ; Runx2 fl / fl (mutant) mice one week after induction at one month of age. (B) Volcano plot revealing that 299 genes were upregulated and 212 genes were downregulated (>2-fold, p < 0.05) in Gli1 - Cre ERT2 ; Runx2 fl / fl mice. (C) IPA based on RNA-seq data. (D) Immunostaining of Igf2 (a and c), p-Igf1r (e and g), p-Irs1 (i and k), and p-Akt (m and o) in incisors from Runx2 fl / fl mice (a, e, i, and m) and Gli1 - Cre ERT2 ; Runx2 fl / fl mice (c, g, k, and o) 1 week after induction at 1 month of age. (b, d, f, h, j, l, n, and p) High-magnification images of the insets in (a), (c), (e), (g), (i), (k), (m), and (o), respectively. Arrows indicate a positive signal, and asterisks indicate the absence of a signal. (E) Western blot of Igf2, p-Igf1r, Igf1r, p-Irs1, Irs1, p-Akt, and Akt in the incisor mesenchyme from Runx2 fl / fl (control) and Gli1 - Cre ERT2 ; Runx2 fl / fl (mutant) mice one week after induction. (F) Igfbp3 RNAscope and Runx2 immunostaining of incisor from Runx2 fl / fl mice (a) and Gli1 - Cre ERT2 ; Runx2 fl / fl mice (c) 1 week after induction at 1 month of age. (b and d) High-magnification images of the insets in (a) and (c), respectively. (e) Real-time PCR analysis of Igfbp3 in the incisor mesenchyme from Runx2 fl / fl and Gli1 - Cre ERT2 ; Runx2 fl / fl mice 1 week after induction at 1 month of age. n = 3/group, *p < 0.05. Arrows indicate coexpression of the Igfbp3 mRNA and Runx2 protein, and asterisks indicate the absence of a signal. (G) ELISA showing the secreted Igf2 in the culture medium supernatant from the MSCs of Runx2 fl / fl (control) and Gli1 - Cre ERT2 ; Runx2 fl / fl (mutant) mice with or without Igfbp3 protein. n = 3, ***p < 0.001. (H) ATAC-seq analysis of the incisor mesenchyme from 1-month-old wild-type mice. The black box indicates the Runx2 binding motif located at the promoter of Igfbp3 . (I) ChIP with Runx2 antibody (or immunoglobulin G [IgG]), followed by qPCR to compare levels of Runx2 the near transcription start site of Igfbp3 , in the incisor mesenchyme from 1-month-old wild-type mice. n = 4, **p < 0.01. The schematic at the bottom of each group of subpanels shows tamoxifen induction protocols. The white dotted line in (D) and (F) shows the cervical loop. All data are represented as mean ± SEM. Scale bars, (D) 50 μm, (F) 100 μm.

Article Snippet: After 80% confluency, the culture plate was washed with Phosphate-buffered saline (PBS) for three times and then added 200 μL serum-free media, i.e., α-MEM with or without 50nM mouse recombinant Igfbp3 protein (R&D Systems, 775-B3).

Techniques: Gene Expression, Control, Mutagenesis, RNA Sequencing, Immunostaining, Western Blot, RNAscope, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Cell reports

Article Title: Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling

doi: 10.1016/j.celrep.2020.108007

Figure Lengend Snippet:

Article Snippet: After 80% confluency, the culture plate was washed with Phosphate-buffered saline (PBS) for three times and then added 200 μL serum-free media, i.e., α-MEM with or without 50nM mouse recombinant Igfbp3 protein (R&D Systems, 775-B3).

Techniques: Recombinant, Protease Inhibitor, TUNEL Assay, In Situ, Imaging, RNAscope, Multiplex Assay, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Software