Fig 1: (A) Flow cytometry profile of untransduced MEFs (UT) and MEFs transduced with the different lentiviruses (EF1GFP, 2AGFP_MOZTIF2, and S8MOZ-TIF2) and therefore expressing GFP. Numbers represent the percentages of cells positive for GFP 48 hours after transduction in each case (n = 3 for each genotype). Multiplicity of infection (MOI) = 30. (B) PCR for the MOZ-TIF2 transcript on untransduced cells and MEFs transduced with the different lentiviruses. (C) Quantitative PCR revealing the relative expression levels of p16INK4a/p19ARF in WT and MozHAT–/– MEFs (passage 3) untransduced or transduced with the different lentiviruses. The transcript levels were normalized to ß-actin for all reactions. Values reflect averages of triplicate samples. Bars represent standard errors of the mean values. (D) Growth curves of cultures of WT and MozHAT–/– MEFs transduced with the different lentiviruses. The graph represents the average values from three independent cultures. Passage numbers are indicated. Bars represent standard errors of the mean values. (E) Flow cytometry profile of untransduced (UT) CD34+cKit+ hematopoietic progenitors and the same cells transduced with either EF1GFP or 2AGFP_MOZTIF2 lentivirus. Numbers represent the percentages of cells positive for GFP 48 hours after transduction in each case. MOI = 50. (F) Specific PCR for the detection of MOZ-TIF2 transcripts in transduced CD34+cKit+ cells. (G) Growth curves of WT CD34+cKit+ cultures transduced with the different lentiviruses. The graph represents the average values from three independent cultures. Passage numbers are indicated. Bars indicate standard errors of the mean values.
Fig 2: Schematic representation of Gm31629 regulating BMSCs senescence and bone regeneration.Gm31629 interacts with YB-1 and delays its degradation, thus decreasing the transcription of p16INK4A and suppressing the senescence of BMSCs. In old subjects, the decreased expression of Gm31629 drives the senescence of BMSCs and leads to impaired bone regeneration.
Fig 3: Gm31629 regulates BMSCs senescence through YB-1/P16INK4A pathway.(A) Western blot analysis of YB-1 pulled-down by Gm31629 (1-1301S) and antisense Gm31629 (1-1301AS) or other controls. (B) YB-1-retrieved Gm31629 RNA as determined by RT-qPCR analysis. The level of IgG-retrieved Gm31629 was set at an arbitrary value = 1. (C) Western blotting analysis of YB-1 protein in WT and Gm31629-KO BMSCs. (D) Western blotting analysis of YB-1 protein in adenovirus vector-driven Gm31629 overexpressed or control BMSCs. (E) Western blotting analysis of YB-1 protein in WT and Gm31629-KO BMSCs treated with CHX. (F) The binding of YB1 to the p16INK4A promoter was detected by ChIP -PCR assay with an antibody against YB1 or IgG. (G) The abundance of YB-1 binding on the promoter of p16INK4A was determined by ChIP assay followed by RT-qPCR analysis. (H) Western blotting result of YB-1 and P16INK4A protein in WT and Gm31629-KO BMSCs with or without YB-1 overexpressed. (I) Representative images of SA-ßGal staining of BMSCs. Scale bar:50 µm.(J) The percentage of SA-ßGal positive cells. (n = 3). (K) ARS staining of BMSCs under osteogenic induction. Scale bar:100 µm. (L) Quantification of calcium mineralization. (n = 3). Data are expressed as mean ± sd and statistical differences were analyzed by Student’s t test or one-way ANOVA. **P < 0.01; ***P < 0.001.
Fig 4: (A) Analysis of p16INK4a/p19ARF transcript levels in cKit+ cells expressing MOZ-TIF2, MOZ-TIF2 with the mutated HAT domain of MOZ, or the control virus (n = 2). (B) Western blot analysis of p16INK4a protein levels in the cells expressing the mutated form of MOZ-TIF2 compared with WT MOZ-TIF2 cells. (C) Photographs of cells expressing either the WT or the mutant MOZ-TIF2 fusion protein and control cells after SA-ß-Gal staining (left) and quantification (right) (n = 3). (D) ChIP analysis of the recruitment of the Ty1-tagged MOZ-TIF2 using an anti-Ty1 antibody (n = 2). (E) Analysis of p53 transcript levels in cKit+ expressing MOZ-TIF2 or the control virus (n = 3). (F) Western blot analysis of p53 and p53 acetylated at lysine 120 (p53K120) protein levels in cells expressing MOZ-TIF2 or control cells. (G) Analysis of p21 transcript levels in cKit+ expressing MOZ-TIF2 or the control virus (n = 3). (H) Flow cytometry analysis of apoptosis using annexin V and 7ADD staining. Results are representative images from two independent experiments.
Fig 5: (A) Schematic representation of the experimental design. Bone marrow cKit+ cells infected with a retrovirus encoding MOZ-TIF2 were tested for their leukemic potential and senescence status. (B) Serial replating of MOZ-TIF2 (MT2)-expressing cells and GFP control cells (n = 3). (C) Photographs of the colonies after the second replating. Representative images from three independent experiments. (D) MGG staining of cytospin from the second replating. Representative images from three independent experiments. (E) Analysis of HOXA9 transcript levels in cKit+ expressing MOZ-TIF2 or the control virus (n = 3). (F) Flow cytometry detection of MOZ-TIF2-expressing cells (GFP+) in the bone marrow (BM) and the spleen of mice culled because of sickness. Representative FACS plots of five mice. (G) Analysis of p16INK4a/p19ARF transcript levels in cKit+ expressing MOZ-TIF2 or cells transduced with the control virus (n = 3). (H) Western blot analysis for p16INK4a protein levels in the MOZ-TIF2 and control cells. Results are representative of two independent experiments. (I) Flow cytometry analysis of cell cycle. (J) Photographs of MT2 and control cells after SA-ß-Gal staining and quantification (n = 3). (K) Analysis of IL6 transcript levels in cKit+ expressing MOZ-TIF2 or in control cells (n = 3).
Supplier Page from MilliporeSigma for Anti-p16 INK antibody produced in rabbit