Fig 2: Efficiency of UMG-lenti vectors in the transduction of primary human CD34+ cells.CD34+ cells purified from cord blood were transduced with FUIGW, UMG-LV6 or UMG-LV11 viruses carrying the cDNAs for 3xFLAG-ZNF521 and EGFP. (A) FACS analysis of the transduced cells 5 days after transduction. The percentages of EGFP positive cells are indicated. (B) Western blotting analysis of FLAG-ZNF521 and EGFP expression was performed as described above on nuclear and cytosolic extracts. HDAC1 was used as a control for the amounts of extract loaded.

Fig 3: RUNX2 is physically associated with the NuRD(MTA1) complex and the CRL4B complex.A Immunoaffinity purification and mass spectrometry analysis of RUNX2-containing protein complexes. Whole-cell extracts from MDA-MB-231 cells stably expressing FLAG-Vector or FLAG-RUNX2 were immunopurified using anti-FLAG affinity columns and eluents with FLAG peptide. Elutes were resolved using SDS-PAGE and silver-stained. Protein bands were retrieved and analyzed using mass spectrometry. B Western blotting analysis of the purified fractions using antibodies against FLAG in MDA-MB-231 cells. C Immunoprecipitation with antibody against RUNX2 followed by immunoblotting with antibodies against MTA1, MTA2, MTA3, HDAC1, HDAC2, RbAp46/48, MBD3, CUL4B, DDB1, and ROC1. D Immunoprecipitation with antibodies against MTA1, MTA2, MTA3, HDAC1, HDAC2, RbAp46/48, MBD3, CUL4B, DDB1, and ROC1 followed by immunoblotting with antibody against RUNX2. E, F GST pull-down assays with GST-fused proteins expressed in bacteria and in vitro translated proteins as indicated. G–I Identification of the essential domains of RUNX2, MTA1, or CUL4B required for interaction. J Schematic diagram depicting molecular interactions among RUNX2/NuRD(MTA1)/CRL4B complex. IB, immunoblotting; aa, amino acid.

Fig 4: UMG-LVs efficiency in non hematopoietic cells.(A) Non-hematopoietic cell lines, HEK293T, MS-5, NIH-3T3 and DAOY, were transduced with FUIGW, FUIGW-ZNF521, UMG-LV6-ZNF521 and UMG-LV11-ZNF521 and analyzed by FACS to assess the percentage of EGFP positive cells. (B) Nuclear and cytosolic extracts were assayed with anti-FLAG and anti-EGFP antibodies as described above. HDAC1 was used as loading control.

Fig 5: Effect of CWHM-12 on PSC activation in cerulein-treated mice. α-SMA was used as a marker of PSC activation. (A) Immunostaining of pancreatic sections with α-SMA–specific antibody shows normal perivascular staining in control pancreas (left), substantial periacinar staining after cerulein treatment (center), and prevention of PSC activation by CWHM-12 administration (right). (B) Representative Western blot of pancreatic extracts with α-SMA–specific antibody confirmed the differences seen histologically. Expression of α-SMA was normalized to the histone deacetylase 1 protein (HDAC1). (C) Densitometric analysis of α-SMA expression. Data are expressed as means ± SEM; n = 5. **P < .01. (D) Vimentin was used as a marker of both quiescent and activated PSCs. Co-immunofluorescent staining of pancreatic sections with vimentin and α-SMA specific antibody was conducted as described in Materials and Methods. Representative picture demonstrates that normal control pancreases do not have expression of vimentin or α-SMA except for the α-SMA expression in smooth muscle cells surrounding blood vessel. After ceruein treatments, most of vimentin expressing PSC are α-SMA positive. (E) Immunostaining of pancreatic sections with vimentin-specific antibody showed high expression in pancreas of cerulein-treated mice, which was decreased partially with CWHM-12 administration. (F) Expression of vimentin mRNA measured by real-time quantitative PCR analysis and normalized to ribosomal protein, large, P0 shows a similar pattern of vimentin expression. Data are shown as the fold increase over the level of the control group of mice (no cerulein treatment) and expressed as means ± SEM; n = 9. ***P < .001, **P < .01. DAPI, 4',6-diamidino-2-phenylindole; Vim, vimentin.