Fig 1: Treg-cell-specific overexpression of Id2 results in systemic autoimmunity in older mice. a A representative picture of 12–16 week-old Foxp3YFP−Cre and Id2EmGFPFoxp3YFP−Cre mice (left) and spleen (SP) and peripheral lymph nodes (pLN) derived from them (right). b Representative hematoxylin and eosin (H&E)-stained skin, lung, liver sections from 12–16 weeks old Foxp3YFP−Cre and Id2EmGFPFoxp3YFP−Cre mice (scale bar, 100 μm). c Representative FACS plots and percentages of CD4+ and CD8+ T cells from SP and pLN of 12–16 weeks old Foxp3YFP−Cre and Id2EmGFPFoxp3YFP−Cre mice. d, e Representative FACS plots and percentages of CD62LhiCD44lo (naive) and CD62LloCD44hi (effector/memory) cells among CD4+ and CD8+ T cells in SP and pLN 12–16 week-old mice. f Representative FACS plots and percentages of Foxp3+ Treg cells in thymus, SP and pLN from 12–16 week-old mice. NS, not significant, *P < 0.05, **P < 0.005 (Student’s t-test). All data are representative three independent experiments (error bars, s.d.)
Fig 2: The expression profile of AAT knockdown related genes(A) Heat-map of genes altered by AAT knockdown. (B) QPCR was performed to measure the mRNA expression of AAT, Id1, Id2, Id3, Id4, Smad2, Smad3 and Smad4 in HPT-8, HUVEC and JEG-3 cells. (C) Western blot was performed to measure the protein expression of AAT, Id1, Id2, Id3, Id4, Smad2, Smad3 and Smad4 in HPT-8, HUVEC and JEG-3 cells. Right panel, Quantification of the bands.
Fig 3: Downstream target genes regulated by Id2 knockdown. mRNA expression levels of (A) FAK, (B) RhoA, (C) ROCK, (D) MLC1, (E) FBXL14, (F) CD44, (G) Twist, and (H) HOXD10 target genes in Id2-knockdown cells and shNC control cells were measured using qRT-PCR. Internal control: GAPDH. Data (means ± SDs) were collected from three independent experiments. *P < 0.05 versus shNC control.
Fig 4: Overexpression of Id2 suppressed highly invasive LADC cells’ aggressive properties. (A) mRNA expression of Id2 in transfected CL1-5 cells measured using qRT-PCR. The control cells (Mock) were pcDNA3.1 vector transfectants. Internal control: TBP. (B) Expression of Id2 protein in the transfectants, measured through Western blot analysis with an antibody against Id2; loading control: GAPDH. *, non-specific band. (C) Representative images of stably expressing Id2 or control vector cells; morphology and immunofluorescence staining of endogenous F-actin. Red arrows indicate the filopodia of cells; scale bar, 20 µm. (D) Scratch wound healing assays for assessing Id2 transfectant cell migratory ability. Eight hours after wound affliction, the cells migrating to the cell-free zone were counted. Data are presented as mean ± SD and represent three independent experiments. *P < 0.05 versus mock control. (E) Transwell assays were used to evaluate mock, Id2, and CL1-5 transfectant invasiveness in three independent experiments. *P < 0.05 versus mock control. (F) Trypan blue exclusion assay for examination of mock, Id2, and CL1-5 transfectant proliferation activity. *P < 0.05 versus mock control. Assays of (G) anchorage-dependent as well as (H) anchorage-independent colony formation were also executed in mock, Id2, and CL1-5 transfectants. *P < 0.05 versus the mock control.
Fig 5: Id2 dependent Treg plasticity enhances antitumor immunity. a Schematic for doxycycline (Dox)-inducible Id2 overexpression in Treg cells in TetR-Id2EmGFPFoxp3YFP-Cre mice. b Id2 expression was assessed by intracellular staining in SP and pLN from both PBS or Dox treated groups. c Experimental scheme of mouse melanoma model. Females, 8–10 week-old, TetR-Id2EmGFPFoxp3YFP-Cre mice were injected subcutaneously with B16.F10 cells. Mice were treated intraperitoneally either with PBS or Dox from day 0–9, every 72 h. d Tumor progression, expressed as mean tumor volume (mm3) in both treatment groups (PBS; n = 8, Dox; n = 8). e Representative tumor size in PBS and Dox treated groups on d 23. f Difference in tumor weight, as measured at end point of analysis (PBS; n = 8, Dox; n = 8). g Flow cytometry analysis of CD4+Foxp3+ T cells in tumor-draining lymph nodes (dLN), non-draining lymph nodes (non-dLN) as well as within tumor infiltrated lymphocytes. h Intracellular staining of cytokines in CD8+ T cells isolated from dLN, non-dLN and tumor infiltrated lymphocytes, stimulated with phorbol myristate acetate (PMA) and ionomycin for 6 h. NS, not significant, *P < 0.05, **P < 0.005, ***P < 0.001 (Student’s t-test). All data are representative of three independent experiments (error bars, s.d.)
Supplier Page from Abcam for Anti-ID2 antibody [4E12G5]