Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: Effect of MTOR inhibition by rapamycin on HCC cell proliferation. Four different HCC cell lines (HepG2, Huh7, Hep3B and SNU3160) were cultured in conditional media (1% FBS) with or without rapamycin (RAPA). Proliferation of Huh7 or Hep3B cells was inhibited by 10 nM rapamycin for 48 h ( A ). Proliferation of Huh7 or Hep3B cells was regulated by rapamycin in a dose-dependent manner (within 0.1~20 nM) ( B ). PROX1 expression was increased by rapamycin in Huh7 or Hep3B cells. The inhibitory effect of rapamycin was confirmed by detecting the phosphorylated form of MTOR (p-MTOR). MTOR and β-actin were detected as quantitative controls ( C ). Relative expression of PROX1 were analyzed image J ( D ). PROX1 expression was also assessed by immunofluorescence microscopy ( E ). Cell nuclei were observed by DAPI staining. D: DMSO-treated cells, R: rapamycin-treated cells. Scale bars represent 100 μm. * p < 0.05; ** p < 0.01, *** p < 0.001.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Inhibition, Cell Culture, Expressing, Immunofluorescence, Microscopy, Staining

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: PROX1 expression was increased by rapamycin in Huh7 or Hep3B cells. PROX1 expression was elevated in Huh7 ( A ) or Hep3B cells ( E ) after 12~36 h of rapamycin treatment. The inhibitory effect of rapamycin was confirmed by detecting the phosphorylated form of MTOR (p-MTOR). MTOR and β-actin were detected as quantitative controls. The relative analysis comparing PROX1 expression with β-actin shows that PROX1 expression was significantly up-regulated by rapamycin in Huh7 ( B ) or Hep3B cells ( F ). Western blot analysis was performed after Huh7 ( C ) or Hep3B cells ( G ) were treated with rapamycin at concentrations of 0.1~20 nM. Rapamycin activity was confirmed by detecting p-MTOR. MTOR and β-actin were measured as quantitative controls. PROX1 expression in Huh7 ( D ) or Hep3B cells ( H ) was analyzed and compared with β-actin. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Expressing, Western Blot, Activity Assay

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: Rapamycin increased the intracellular half-life of PROX1 protein. The mRNA expression of PROX1 was analyzed by semi-quantitative RT-PCR using total RNAs prepared from Huh7 ( A ) or Hep3B cells ( E ) treated with or without rapamycin. Quantitative RT-PCR results showed that the expression of PROX1 mRNA in Huh7 ( B ) or Hep3B cells ( F ) was not affected by rapamycin. The half-life of PROX1 protein in Huh7 ( C ) or Hep3B cells ( G ) was measured by treating cells with or without rapamycin for 24 h and then exposing them to cycloheximide (50 µg/mL). Phosphorylated MTOR (p-MTOR) was detected to monitor the inhibitory effect of rapamycin. MTOR and β-actin were measured as quantitative controls. Relative analysis showed that PROX1 expression in Huh7 ( D ) or Hep3B cells ( H ) began to decrease after 2 h of cycloheximide treatment in DMSO-treated control cells, but it was maintained after rapamycin treatment for up to 6 h. ** p < 0.01. DMSO: DMSO-treated cells, RAPA: rapamycin-treated cells.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Cells

Article Title: PROX1, a Key Mediator of the Anti-Proliferative Effect of Rapamycin on Hepatocellular Carcinoma Cells

doi: 10.3390/cells11030446

Figure Lengend Snippet: PROX1 played a key role in the anti-proliferative effect of rapamycin. Huh7 ( A ) or Hep3B cells ( E ) were transfected with a plasmid expressing human PROX1 or an empty plasmid, and 24 h later were treated with or without rapamycin for an additional 24 h. Western blot analysis showed that PROX1 expression was increased by the over-expression system and by rapamycin, and markedly by rapamycin treatment plus PROX1 over-expression. D: cells treated with DMSO, R: cells treated with rapamycin. The effect of PROX1 on the proliferation of Huh7 ( B ) or Hep3B cells ( F ) was also investigated using the over-expression system. After 24 h of transfection, cells were treated with or without rapamycin for an additional 48 h. Cell proliferation was inhibited by rapamycin and by PROX1 over-expression. Huh7 ( C ) or Hep3B cells ( G ) were transfected with siRNA targeting PROX1 mRNA (siPROX1) or control siRNA (siCTR). After 24 h, cells were treated with or without rapamycin for an additional 24 h. Western blot analysis clearly showed the knock-down effect of siPROX1. The inhibitory effect of rapamycin was confirmed by determining phosphorylated MTOR (p-MTOR). MTOR and β-actin were detected as quantitative controls. To investigate the effect of siPROX1 on cell proliferation, Huh7 ( D ) or Hep3B cells ( H ) were transfected with siPROX1 and treated with or without rapamycin for an additional 48 h. siPROX1treatment was found to increase the proliferation of Huh7 or Hep3B cells and to decrease the anti-proliferative effect of rapamycin. **, p < 0.01; ***, p < 0.001. DMSO: DMSO-treated cells, RAPA: rapamycin-treated cells.

Article Snippet: For Western blot assay, 20 μg of proteins was separated by 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (GE HealthCare, Hatfield, UK), which were then treated with anti-PROX1 antibody [ ], anti-phospho-MTOR (p-MTOR, ser-2448) antibody (sc-101738, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MTOR antibody (Ab-2448, Cusabio, Houston, TX, USA), anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, CA), appropriate secondary antibodies, and enhanced chemiluminescence detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Over Expression, Control, Knockdown

Journal:

Article Title: Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs

doi: 10.1073/pnas.0401901101

Figure Lengend Snippet: IL-6Rα is synthesized from constitutive mRNA by human PMNs in response to inflammatory signaling to mTOR. PMNs were incubated with control buffer, PAF, or other inflammatory agonists in suspension, and IL-6Rα protein was detected by Western analysis with the same antibody used for immunocytochemical studies (Fig. 3). (A) PMNs were incubated with control buffer (lane C) or increasing concentrations of PAF for 1 h. (B) PMNs were incubated with control buffer, 100 nM PAF, PAF that had been preincubated with recombinant human PAF acetylhydrolase (rPAF-AH), or the indicated agonists. (C) HUVEC monolayers were incubated with PMNs activated with 100 nM PAF alone or after pretreatment with rapamycin by using separated chambers and were then blotted for IL-6Rα. Control HUVEC were incubated in the absence of PAF-stimulated PMNs. Lane 1, control HUVEC; lane 2, HUVEC incubated with PMNs pretreated with rapamycin before stimulation with PAF; lane 3, HUVEC incubated with PAF-stimulated PMNs. (D) PMNs were pretreated with control buffer or actinomycin D followed by activation with 100 nM PAF for the times shown. Blots from PMNs activated with PAF alone or after pretreatment with actinomycin D are shown above and below one another at each time point. Control PMNs were lysed in the absence of actinomycin D or PAF and blotted (leftmost lane in Upper). In some experiments IL-6Rα stains as a doublet or triplet on immunoblotting, as shown. (E) PMNs were pretreated with control buffer or rapamycin and then activated with 100 nM PAF for the times indicated. In a control experiment, neither actinomycin D nor rapamycin alone in the absence of PAF induced an IL-6Rα band (data not shown).

Article Snippet: Antibodies against IL-6Rα (rabbit polyclonal anti-IL-6R, sc-661, C-2, Santa Cruz Biotechnology) or mTOR (rabbit polyclonal anti-mTOR, KAP-P1002, StressGen Biotechnologies, Victoria, BC, Canada) were incubated with the cells overnight in HBSS/5.0% goat serum at 4°C.

Techniques: Synthesized, Incubation, Western Blot, Recombinant, Activation Assay

Journal:

Article Title: Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs

doi: 10.1073/pnas.0401901101

Figure Lengend Snippet: The mTOR pathway regulates signal-dependent translation in human neutrophils activated via the PAF receptor. (A) Freshly isolated PMNs were activated with 100 nM PAF for 30 min followed by staining for mTOR. (B) The peptide immunogen against which the anti-mTOR antibody was raised completely quenched the immunofluorescent staining. (C) Components of the mTOR pathway in human neutrophils activated by PAF (also see Fig. 6). S6K1 and related members of this family are also called p70S6 kinases. In other cell types, phosphatidylinositol 3-kinase (PI-3K) lies upstream of mTOR and may also mediate parallel signaling to S6K1 and 4E-BP1 (20), but this has not been formally demonstrated for signaling to the mTOR pathway via the PAF receptor.

Article Snippet: Antibodies against IL-6Rα (rabbit polyclonal anti-IL-6R, sc-661, C-2, Santa Cruz Biotechnology) or mTOR (rabbit polyclonal anti-mTOR, KAP-P1002, StressGen Biotechnologies, Victoria, BC, Canada) were incubated with the cells overnight in HBSS/5.0% goat serum at 4°C.

Techniques: Isolation, Staining