(A–C) Overexpression of CHIP results in the facilitated differentiation of MC3T3-E1 cells into adipocytes

MC3T3-E1-CHIP cell lines (#2 and #8) and the mock cells were cultured in the presence or absence of DIM mixture for 7 or 14 d. Accumulation of cytoplasmic triglyceride was detected by Oil Red O staining (A). A quantitative representation of positive Oil Red O staining adipocytes. Cell numbers were counted on three randomized fields with a phase-contrast microscope. The percentages of triglyceride-containing cells are shown with mean and SD (error bars; B). The overexpression of CHIP induces the expression of adipocyte marker genes PPARγ and CEBPα. RT-PCR was performed on day 7 (C). (D) Establishment of cell lines stably expressing HA-CHIP in C3H10T1/2 cells (#8 and #11). Protein levels of the endogenous CHIP and HA-CHIP are shown. (E–G) CHIP does not affect the ability of C3H10T1/2 cells to differentiate into adipocytes. C3H10T1/2-CHIP cell lines (#8 and #11) were used in adipocyte differentiation experiments. Oil Red O staining shows accumulation of cytoplasmic triglyceride on day 14 (E) with a quantitative representation of positive Oil Red O staining adipocytes (F) and expression of PPARγ and CEBPα on day 7 (G).<p><b>Copyright information:</b></p><p>Taken from "CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation"</p><p></p><p>The Journal of Cell Biology 2008;181(6):959-972.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426947.</p><p></p

(A) Establishment of cell lines stably expressing HA-CHIP in MC3T3-E1 cells (2 and 8)

Endogenous CHIP and HA-CHIP expression was measured by immunoblotting with an anti-CHIP antiserum. (B) Stable expression of HA-CHIP in MC3T3-E1 cells reduces the endogenous Runx2 protein levels. The cell lines were treated with AA/β-GP for the indicated days. The mRNA (top) and protein (bottom) levels of Runx2 were measured by RT-PCR and Western blotting, respectively. (C) Stable depletion of CHIP using siRNA in MC3T3-E1 cells increased Runx2 protein levels. The cell lines with depletion of CHIP (#9 and #20) were treated with AA/β-GP for the indicated days. mRNA (left) and protein (right) levels of Runx2 were measured by RT-PCR and Western blotting, respectively. (D–F) CHIP inhibits the differentiation of MC3T3-E1 cells into osteoblast-like cells. CHIP- or CHIP siRNA–expressing cell lines were induced to differentiate into osteoblast-like cells in the presence of AA/β-GP for 4–21 d followed by the determination of AP activity (D), calcium accumulation by Alizarin red staining (E), and expression of the osteoblast marker genes (BSP and OCN) by real-time RT-PCR (F). Error bars represent SEM.<p><b>Copyright information:</b></p><p>Taken from "CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation"</p><p></p><p>The Journal of Cell Biology 2008;181(6):959-972.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426947.</p><p></p

(A) Overexpression of CHIP inhibits Runx2-induced transcriptional activity

Luciferase assays were performed using NIH3T3 or COS7 cells transfected with the indicated expression vectors along with a Runx2-responsive reporter construct, 6xOSE2-OC/pGL3 and pRL-TK (internal control). Values were normalized using the internal control and presented relative to basal activity (NIH3T3, first bar; COS7, fifth bar) with the mean from three independent repeats. (B) Mutants of CHIP fail to inhibit Runx2-induced transcriptional activity. CHIP, CHIP mutants, and Flag-Smurf1 were compared for the inhibitory effect on Runx2 activity in NIH3T3 cells. (C) Knocking down CHIP by siRNA facilitates Runx2-mediated transcription in UMR106 cells. Luciferase assays were performed in UMR106 cells transfected with Runx2 in the presence of CHIP, EGFP RNAi, or CHIP RNAi. (D) CHIP did not affect the transcriptional activity of STAT3. NIH3T3 cells transfected with STAT3 in the presence or absence of CHIP along with pGL3–acute phase response element and treated with 10 ng/ml interleukin-6. All of the data are presented as mean with SEM (error bars) from three independent experiments. The same letter represents a significant difference within this group in a meaningful comparison (P < 0.05).<p><b>Copyright information:</b></p><p>Taken from "CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation"</p><p></p><p>The Journal of Cell Biology 2008;181(6):959-972.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426947.</p><p></p

(A) von Kossa staining for MC3T3-E1 cells cultured for 3 wk in the presence of AA/β-GP was performed to demonstrate the cellular phenotype of osteoblasts

(B) mRNA and protein levels of Runx2 and CHIP during the osteoblast differentiation were compared. RT-PCR and Western blot (see quantitative presentations in Fig. S2, available at ) analyses were performed on MC3T3-E1 cells induced by AA/β-GP for the indicated number of days. (C) Smurf1 expression is unchanged, and WWP1/Shn3 is increased during osteoblast differentiation. Real-time RT-PCR analysis was performed for the expression of the indicated genes during osteoblast differentiation of MC3T3-E1 cells. Values are shown relative to cells on day 0. (D) Alizarin red staining for mouse calvarial cells cultured for 2 wk in the presence of AA/β-GP. (E and F) The expression of Runx2, CHIP, WWP1, Shn3, and Smurf1 in mouse calvarial cells was analyzed as in B and C. (G) Real-time RT-PCR was performed to show expression of the indicated genes in different lineages of mesenchyma-originated cells. Fold induction was calculated based on the mRNA level of each gene in NIH3T3. (H) Expression of the CHIP (green) and Runx2 (red) proteins in osteoblasts in vivo. Double immunofluorescence staining was performed using sections of trabecular (femur; a and b) and calvarial (c and d) bone from newborn mice. Enlarged osteoblasts (boxed) are shown on the bottom (b and d). Ob, osteoblast; Chon, chondrocyte; Fi, fibroblast-like or other types of cells. Error bars represent SEM.<p><b>Copyright information:</b></p><p>Taken from "CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation"</p><p></p><p>The Journal of Cell Biology 2008;181(6):959-972.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426947.</p><p></p

Functional Domains of Androgen Receptor Coactivator p44/Mep50/WDR77and Its Interaction with Smad1

<div><p>p44/MEP50/WDR77 has been identified as a coactivator of androgen receptor (AR), with distinct growth suppression and promotion function in gender specific endocrine organs and their malignancies. We dissected the functional domains of p44 for protein interaction with transcription factors, transcriptional activation, as well as the functional domains in p44 related to its growth inhibition in prostate cancer. Using a yeast two-hybrid screen, we identified a novel transcription complex AR-p44-Smad1, confirmed for physical interaction by co-immunoprecipitaion and functional interaction with luciferase assays in human prostate cancer cells. Yeast two-hybrid assay revealed that the N-terminal region of p44, instead of the traditional WD40 domain at the C-terminus, mediates the interaction among p44, N-terminus of AR and full length Smad1. Although both N and C terminal domains of p44 are necessary for maximum AR transcriptional activation, the N terminal fragment of p44 alone maintains the basic effect on AR transcriptional activation. Cell proliferation assays with N- and C- terminal deletion mutations indicated that the central portion of p44 is required for nuclear p44 mediated prostate cancer growth inhibition.</p></div