SHP protein is localized on the mitochondrial outer membrane.

<p>(A) Western blots to detect Flag-mSHP, VADC, COXIV, HSP60, and Cyto C proteins using specific antibodies for each protein, with the exception of SHP, which was detected with an anti-Flag antibody. Huh7 cells were transfected with Flag-mSHP (20 µg, 15 cm plate), and five plates were used to harvest protein. Differential centrifugation was used to isolate the mitochondria-enriched precipitate fraction (P1), microsome-enriched precipitate fraction (P2), and cytosolic supernatant fraction (S); the whole cell lysate (W) was used for comparison. (B) Alkaline digestion of mitochondrial membrane fraction (P^m) and soluble fraction (S^m) and Western blots to determine Flag-mSHP, VADC, COXIV, HSP60, and Cyto C proteins. (C) Trypsin protection assays and Western blots to determine Flag-mSHP, VADC, Bcl2, and Cyto C proteins in mitochondria. Abbreviations: mito, mitochondria; mem, membrane; solu, soluble; peri, peripheral; pt, protein.</p

Ectopic expression of HNF4α induces an exclusive SHP nuclear translocation.

<p>(A) The subcellular localization of GFP-hSHP in 293T cells was observed under a fluorescence microscope, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 100 µm, 50 µm (insets). (B) The N-terminal domain of SHP is primarily responsible for the interaction with HNF4α protein. Left, Diagram showing the deletion mutation constructs of GFP-mShp. Right, 293T cells were transfected with GFP-mSHP deletion constructs (4 µg, 6 cm plate) along with HA-HNF4α (4 µg, 6 cm plate) expression vector. Anti-HA agarose was used to immuneprecipitate HNF4α, and the protein levels of various GFP-mSHP truncation mutants and HNF4α were detected by Western blots using anti-GFP or anti-HA antibodies, respectively. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.</p

Deletion of the N-terminal fragment increases SHP protein levels in mitochondria.

<p>(A) Diagram showing the deletion mutation constructs of Flag-hSHP. FL, full length; ND, N-terminal deletion. (B) Left, Western blots to determine SHP protein expression in different cellular fractions in Huh7 cells. Right, band intensities were measured by densitometry, and the intensities relative to that of the whole cell lysate (set as 1) were plotted. Data are represented as mean ± SEM (*, <i>P</i><0.01). Abbreviations: whole, whole cell lysate protein; nu, nuclear protein; mito, mitochondria protein. PARP is a marker for nuclear proteins; VDAC is expressed in mitochondria.</p

Overexpression of miR-127 inhibits MMP13 and TGFβ-mediated induction of HCC cell migration.

<p>(<i>A</i>) Cell migration assay. MHCC97H cells were transfected with MMP13 siRNA (siMMP13) (20 nM) in the absence or presence of pTarget or pTarget-miR-127 (2 µg). After cells were seeded onto the inserts, lower chamber media were treated with MMP13 peptide (50 ng/ml) as indicated. Cells that had migrated through the membrane were fixed and stained with crystal violet (left). Quantitative results on the right. (<i>B-C</i>) qPCR analysis of MMP13 (<i>B</i>) and miR-127 (<i>C</i>) expression under the same experimental conditions as (<i>A</i>). (<i>D</i>) Cell migration assay. MHCC97H and Hepa-1 cells were transfected with pTarget or pTarget-miR-127 (2 µg), in the absence or presence of TGFβ (5 ng/ml). Migrated cells were stained with crystal violet and visualized by microscopy (left). Quantitative results on the right. (<i>A-D</i>): *p<0.01, miR-127 <i>vs.</i> pTarget in control group; ^¥p<0.01, pTarget in MMP13 group <i>vs.</i> pTarget in control group;^‡p<0.01, miR-127 <i>vs.</i> pTarget in MMP13 group; ^§p<0.01, miR-127 <i>vs.</i> pTarget in siMMP13 group.</p

Model of negative-feedback regulation of miR-127 expression by TGFβ/c-Jun that controls MMP13 expression and stability in HCC.

<p>TGFβ activates the oncogene c-Jun through ERK and JNK pathways. The activation of c-Jun serves a dual function, which involves induction of MMP13 gene expression but repression of miR-127 gene transcription by inhibiting miR-127 promoter activity. c-Jun also antagonizes p53 activation of the miR-127 promoter and gene transcription. On the other hand, overexpression of miR-127 decreases MMP13 protein levels by binding to its 3′UTR and causing MMP13 degradation, thus diminishing TGFβ-mediated HCC migration.</p

Overexpression of miR-127 inhibits HCC cell migration and tumor growth.

<p>(<i>A</i>) Wound healing assay. MHCC97H cells were transfected with either an empty vector pTarget (-) or a miR-127 expression vector in pTarget (+) (2 µg). Wound closure was photographed 48 hr later (left) and the residual gap between the migrating cells was quantified (right), which is expressed as a percentage of the initial scraped area. (<i>B</i>) Cell migration assay. MHCC97H cells were transfected with pTarget or pTarget-miR127 (2 µg), a negative (neg) control or miR-127 inhibitor (anti-miR-127) (20 nM). Cells that had migrated through the membrane of Transwell inserts were fixed, stained with crystal violet, and visualized by microscopy. (<i>C</i>) Cell invasion assay. MHCC97H cells were transfected with pTarget or pTarget-miR127 (2 µg), and seeded onto pre-coated inserts with BME solution and incubated for 16 hr. The invaded cells were dissolved in Cell Dissociation Solution/Calcein-AM and absorbance was measured. (<i>D</i>) Tumorigenesis assay. Control 97HLuc-Sico or 97HLuc-miR-127 cells were grafted subcutaneously in the dorsa of athymic mice, and tumor growth was monitored using the Xenogen bioluminescent imaging system by day 9. The number represents tumor images detected (photons/s/cm²/steridian). *p<0.01, miR-127 <i>vs.</i> pTarget control; ^¥p<0.01, anti-miR-127 <i>vs.</i> negative control.</p

TGFβ enhances c-Jun-mediated repression of miR-127 via ERK and JNK pathways.

<p>(<i>A-C</i>) qPCR analysis of c-Jun (<i>A</i>), miR-127 (<i>B</i>), and MMP13 (<i>C</i>) mRNA expression in MHCC97H cells treated with TGFβR inhibitor (SB 431542, 10 µM), NFκB inhibitor (BAY-11-7082, 5 µM, ERK inhibitor (PD 98059, 50 µM), p38 inhibitor (SB 203580, 10 µM) and JNK inhibitor (SP 600125, 50 µM),) in the absence (−) or presence of TGFβ (+) (5 ng/ml). Statistical results represent the mean ± SD. *p<0.01, TGFβ (+) <i>vs.</i> TGFβ (−) group.</p

TGFβ represses miR-127 expression by enhancing c-Jun activity.

<p>(<i>A</i>) qPCR analysis of MMP13 (left), c-Jun (middle), and miR-127 (right) expression in MHCC97H cells treated with TGFβ. *p<0.01, TGFβ (+) <i>vs.</i> TGFβ (-) group. (<i>B</i>) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter (pro) luciferase (luc) reporter, and c-Jun or c-Fos expression plasmids. *p<0.01, c-Jun (+) <i>vs.</i> c-Jun (-) group. (<i>C)</i> Transient transfection assay. Hela cells were co-transfected with miR-127 or AP1 promoter reporter along with c-Jun/c-Fos plasmid (100 ng) in the absence or presence of TGFβ (5 ng/ml). *p<0.01, c-Jun/Fos <i>vs.</i> control without (-) or with (+) TGFβ; ^¥p<0.01, control with (+) TGFβ <i>vs.</i> control without (-) TGFβ. (<i>D</i>) Transient transfection assay. Hela cells were co-transfected with miR-127 promoter reporter, c-Jun (100 ng) and/or p53 plasmids (100 ng). *p<0.01, c-Jun or p53 <i>vs.</i> control; ^¥p<0.01, c-Jun/p53 <i>vs.</i> p53. (<i>B-D</i>) Luciferase (Luc) activity was normalized by Renilla activity (act). (<i>E</i>) qPCR analysis of miR-127 and p53 expression in HepG2 cells transfected with control (con) or p53 siRNAs (si-p53) (20 nM), or in HCT116<i>p53^+/+</i> (+) and HCT116<i>p53^−/−</i> (-) cells. *p<0.01, si-p53 <i>vs.</i> control. (<i>F</i>) qPCR analysis of miR-127 expression in MHCC97H cells that were overexpressed with c-Jun and/or p53. *p<0.01, p53 <i>vs.</i> control (-); ^¥p<0.01, c-Jun/p53 <i>vs.</i> p53.</p

miR-127 is downregulated in a subset of HCC specimens.

<p>(<i>A-B</i>) qPCR analysis of miR-127 expression (<i>A</i>) and MMP13 mRNA (<i>B</i>, left), and Western blot (WB) analysis of MMP13 protein (<i>B</i>, right) in 5 pairs of surrounding controls and HCC specimens. *p<0.01, tumor <i>vs.</i> non-tumor.</p

SHP destabilizes p53 protein by enhancing the activity of Mdm2.

<p>A: Western blots. Plasmids expressing Flag-p53 were co-transfected with Flag-SHP plasmids in 293T or Hela cells. p53 or SHP protein levels were determined using anti-Flag antibodies. B: Western blots. Flag-SHP and Myc-p53 plasmids were cotransfected in Hela cells, and SHP and p53 proteins were detected using anti-Flag and anti-Myc antibodies, respectively. C: Western blots. Myc-p53, Flag-SHP or HA-Mdm2 expression vectors were transfected in Hela cells, and Mdm2, SHP and p53 proteins were detected using anti-HA, anti-Flag or anti-Myc antibodies, respectively. D: Ubiquitination assays. Hela cells were cotransfected with GFP-p53, HA-Mdm2, and various Flag-SHP expression vectors together with Myc-ubiquitin (Myc-Ub) plasmids. GFP-p53 was immunoprecipitated from cell extracts with anti-GFP antibodies, and ubiquitinated p53 in the immunoprecipitates was detected by Western blots with anti-Myc antibodies. Positions of ubiquitinated p53 proteins are indicated by a solid line. E: Western blots. HA-Mdm2, Flag-p53 or Flag-SHP expression vectors were transfected in 293T cells, and Mdm2, SHP and p53 proteins were detected using anti-HA or anti-Flag antibodies, respectively. F: Ubiquitination assays. 293T cells were cotransfected with Flag-p53 and GFP-SHP expression vectors together with HA-ubiquitin (HA-Ub) plasmids. Flag-p53 was immunoprecipitated from cell extracts with anti-Flag antibodies, and ubiquitinated p53 in the immunoprecipitates was detected by Western blots with anti-HA antibodies. Positions of ubiquitinated p53 proteins are indicated by a solid line. G: Ubiquitination assays. 293T cells were cotransfected with GFP-p53, HA-Mdm2, and various Flag-SHP expression vectors together with Myc-ubiquitin (Myc-Ub) plasmids. GFP-p53 was immunoprecipitated from cell extracts with anti-GFP antibodies, and ubiquitinated p53 in the immunoprecipitates was detected by Western blots with anti-Myc antibodies. Positions of ubiquitinated p53 proteins are indicated by a solid line.</p