Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 3 c-Met regulates CD44s expression through AKT signaling. (A) MHCC97-H cells were treated with 1 μM of PHA665752 for 24 h. Immunoblot analysis of CD44v6 (~160 kDa), CD44s (~85 kDa), and B-actin. (B) MHCC97-H cells were treated with 25 μM DMSO, LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) for 24 h, and immunoblot analysis was performed. (C) MHCC97-H cells were stably transfected with c-Met shRNA. Lysates were collected after 2 passages and CD44s, fibronectin, and E-cadherin expression was via immunoblotting. (D) Tumorsphere formation (40X magnification) assay of stably transfected MHCC97-H cells with c-Met shRNA compared to scrambled control. The data represent the mean ± SEM of triplicates, *p < 0.01. Phase-contrast images are representative of triplicates (40X magnification).

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, shRNA, Control

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 4 CD44s regulates mesenchymal and tumor-initiating stem-like characteristics. (A) Tumorsphere assay of MHCC97-H cells treated with DMSO, LY294002 or PD98059 (25 μM) for 24 h followed by trypan blue exclusion. 1x10^5 cells were plated in triplicates in 6-well low adherent plates for an additional 2 weeks. The data represent the mean ± SEM of triplicates, *p < 0.05. Phase contrast images are at 40X magnification. (B) Endogenous protein levels of CD44s and the mesenchymal markers E-cadherin and Fibronectin in two pooled stable CD44s shRNA cell lines. (C-D) Heatmap of MHCC97-H CD44s shRNA compared to scrambled control cells. Relative mRNA expression of stem cell genes in CD44s shRNA #1 compared with the scrambled control normalized to GAPDH. The data represent the mean ± SEM of triplicates, *p < 0.001.

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: shRNA, Control, Expressing

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 5 c-Met induces tumor-initiating characteristics through CD44s. (A) 5x104 MHCC97-H cells were plated onto 6-well low adherent culture plates in triplicates. Tumorspheres were collected by centrifugation after 2 weeks . For monolayer cultured cells, 5x104 MHCC97-H were plated on 10 cm plate and cultured for 2 weeks. Media was changed every 2–3 days. MHCC97-H cells were plated in low-adherent cell or monolayer cell culture dishes for two weeks and immunoblotting analysis was confirmed on tumorsphere lysates. The data are representative of three independent experiments. (B) 5x103 MHCC97-H scrambled, c-Met shRNA and CD44s shRNA cells were grown in 6-well low-adherent culture plates for two weeks and the numbers of tumorspheres were counted (40X magnification). The data are representative of two independent experiments and are shown as the mean ± SEM of triplicate plates. (C) CD44s recovers tumorsphere formation. MHCC97-H cells were transfected with c-Met siRNA (25pM) for 24 hrs followed by overexpression of CD44s or pBabe empty vector retrovirus for an additional 48 h (immunoblot) or two weeks (tumorsphere assay). (D) Immunoblot data are representative of two independent experiments. The data for the tumorsphere assay data are representative of two independent experiments and are shown as the mean ± SEM of triplicate wells (40X magnification).

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: Centrifugation, Cell Culture, Western Blot, shRNA, Transfection, Over Expression, Plasmid Preparation

Journal: BMC cancer

Article Title: Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

doi: 10.1186/s12885-015-1166-4

Figure Lengend Snippet: Figure 6 CD44s regulates tumor initiation in vivo. (A) Immunoblot of MHCC97-H scrambled and CD44s shRNA #1 before cells were injected into athymic nude mice. (B-D) Tumor initiation graph of MHCC97-H CD44s shRNA compared with the scrambled shRNA control. Bilateral subcutaneous injections of 1x102, 1x103, or 1x104 cells were inoculated into athymic nude mice, and the number of tumors formed and the percent tumor initiation were calculated (1x102, N = 10; 1x103, N = 8; or 1x104, N = 6). Tumor volume was calculated at the end of the experiment and. Data represent the mean ± SD. Confirmation of down-regulation of CD44s and c-Met signaling was performed by immunoblotting.

Article Snippet: Stable shRNA: TG320418 HuSH 29mer shRNA constructs against c-Met in the pGFP-V-RS vector was purchase from OriGene (Rockville, MD).

Techniques: In Vivo, Western Blot, shRNA, Injection, Control

Journal: Arthritis and rheumatism

Article Title: Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

doi: 10.1002/art.22713

Figure Lengend Snippet: Figure 2. Levels of cytokines in bronchoalveolar lavage (BAL) fluid from Caucasian and African American patients with systemic sclerosis (SSc) and normal controls. Cytokine levels were significantly elevated in BAL fluid from African American control subjects as compared with Caucasian control subjects. A, Levels of hepatocyte growth factor (HGF), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF) were measured by cytokine array analysis. Array images were scanned and analyzed with National Institutes of Health Image software. Positive controls (6 per membrane) were used to normalize the results from the different membranes being compared. Values are the mean and SEM of 24 separate arrays of BAL fluid samples from controls and 40 separate arrays from SSc patients. Variation between 2 identical cytokine spots ranged from 0% to 10% in duplicate experiments. B, Effect of selected cytokines on type I collagen expression in lung fibroblasts from Caucasian and African American normal subjects and SSc patients. Confluent cultures of lung fibroblasts were serum-starved for 24 hours and then incubated for 72 hours with recombinant transforming growth factor (TGF; 5 ng/ml), which was used as a positive control in studies of normal lung fibroblasts, or with HGF, IGFBP-3, OPG, SCF, TPO, or VEGF (50 ng/ml each). Serum-free medium (SFM) was used as a basal level for type I collagen. Cells were collected with lysis buffer and analyzed by Western blotting using anti–type I collagen antibody. Anti–-actin antibody was used as the sample loading control. Shown are representative immunoblots from 3 independent experiments.

Article Snippet: Confluent cultures of lung fibroblasts were serum-starved for 24 hours, followed by incubation for 72 hours with recombinant transforming growth factor (TGF ; 5 ng/ml) and/or HGF (50 ng/ml or concentrations indicated below), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF), with or without neutralizing anti-human HGF receptor (c-Met) antibody (all purchased from R&D Systems).

Techniques: Control, Binding Assay, Software, Membrane, Expressing, Incubation, Recombinant, Positive Control, Lysis, Western Blot

Journal: Arthritis and rheumatism

Article Title: Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

doi: 10.1002/art.22713

Figure Lengend Snippet: Figure 5. Modulation of the effects of hepatocyte growth factor (HGF) on connective tissue growth factor (CTGF) expression and type I collagen accumulation by anti–c-Met antibody treatment of lung fibroblasts from Caucasian patients with systemic sclerosis (SSc) and normal controls. Anti–c-Met antibody (Ab) diminished the effects of HGF on CTGF expression and collagen accumulation. Serum-starved lung fibroblasts were incubated for 72 hours with medium containing anti–c-Met antibody (2 g/ml), with or without HGF (50 ng/ml). Normal lung fibroblasts were left untreated or were additionally treated with transforming growth factor (TGF; 5 ng/ml). Anti–- actin antibody was used as the sample loading control. Shown are representative immunoblots from 3 independent experiments.

Article Snippet: Confluent cultures of lung fibroblasts were serum-starved for 24 hours, followed by incubation for 72 hours with recombinant transforming growth factor (TGF ; 5 ng/ml) and/or HGF (50 ng/ml or concentrations indicated below), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF), with or without neutralizing anti-human HGF receptor (c-Met) antibody (all purchased from R&D Systems).

Techniques: Expressing, Incubation, Control, Western Blot

Journal: Arthritis and rheumatism

Article Title: Impairment of the antifibrotic effect of hepatocyte growth factor in lung fibroblasts from African Americans: possible role in systemic sclerosis.

doi: 10.1002/art.22713

Figure Lengend Snippet: Figure 6. Hepatocyte growth factor (HGF)–induced c-Met receptor phosphorylation in lung fibroblasts from Caucasian and African American patients with systemic sclerosis (SSc). Lung fibroblasts were cultured to confluence on 6-well plates and collected with lysis buffer. Twenty micrograms of protein per sample was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4–20% gradient gels) and analyzed by Western blotting using anti–phospho–c-Met (Ph-C-Met) or anti–c-Met polyclonal antibodies (see Patients and Methods for details). Anti–-actin antibody was used as the sample loading control. Shown are representative immunoblots from 3 independent experiments. A, Levels of phosphor- ylated and total c-Met receptor in lung fibroblasts from Caucasian and African American SSc patients. B, Concentration-dependent phosphorylation of c-Met receptor in lung fibroblasts from Caucasian and African American SSc patients treated with HGF for 5 minutes at the indicated concentrations. C, Time course of c-Met receptor phosphorylation in lung fibroblasts from Caucasian and African American SSc patients after treatment with HGF (50 ng/ml) for various periods (left). Western blots from the time course experiments were analyzed by scanning densitometry using National Institutes of Health Image software (right). Values are the mean of 4 samples at each time point.

Article Snippet: Confluent cultures of lung fibroblasts were serum-starved for 24 hours, followed by incubation for 72 hours with recombinant transforming growth factor (TGF ; 5 ng/ml) and/or HGF (50 ng/ml or concentrations indicated below), insulin-like growth factor binding protein 3 (IGFBP-3), osteoprotegerin (OPG), stem cell factor (SCF), thrombopoietin (TPO), and vascular endothelial growth factor (VEGF), with or without neutralizing anti-human HGF receptor (c-Met) antibody (all purchased from R&D Systems).

Techniques: Phospho-proteomics, Cell Culture, Lysis, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Concentration Assay, Software