Journal: Cancer science

Article Title: Oxysterol binding protein-related protein-5 is related to invasion and poor prognosis in pancreatic cancer.

doi: 10.1111/j.1349-7006.2008.00987.x

Figure Lengend Snippet: Fig. 2. Expression levels of oxysterol binding protein-related protein (ORP)-5 in hamster and human pancreatic cancer cell lines. PC1 was hamster pancreatic cancer cell line with a low potential for invasion and metastasis, and PC1.0 was hamster pancreatic cancer cell line with a high potential for invasion and metastasis. (a) ORP5 expression in the hamster pancreatic cancer cell lines PC1.0 and PC1 after transfection of short interfering RNA (siRNA) or expression vector. ORP5 was expressed at a high level in the PC1.0 cells and at a low level in the PC1 cells, at both the mRNA and protein levels. Transfection of ORP5 siRNA into PC1.0 cells resulted in a significant decrease in the expression level of ORP5 at 48–72 h after transfection. Transfection of pcDNA/hamORP5 into PC1 cells resulted in a significant increase in the expression level of ORP5 at 24–72 h after transfection. (b) The expression levels of ORP5 in human pancreatic cancer cell lines. At both the mRNA and protein level, ORP5 was expressed at a high level in Capan1, Capan2, and Panc1 cells, at a moderate level in the MiaPaCa2 cells, and at a low level in the Hs700T cells. (c) ORP5 expression in the human pancreatic cancer cell lines Capan2 and Hs700T after transfection of siRNA or expression vector. ORP5 was expressed at a high level in the Capan2 cells, but at a low level in the Hs700T cells, at both the mRNA and protein levels. Transfection of ORP5 siRNA into Capan2 cells resulted in a significant decrease in the expression level of ORP5 at 48–72 h after transfection. Transfection of pcDNA/huORP5 into Hs700T cells resulted in a significant increase in the expression level of ORP5 at 24–72 h after transfection. (d) The ORP5 stable transfectant cells (PC1 + ORP5 or Hs700T + ORP5) showed high expression levels of ORP5, and the LacZ stable transfectant cells (PC1 + LacZ or Hs700T + LacZ) showed low expression levels of ORP5. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription–polymerase chain reaction.

Article Snippet: The membrane was blocked with 5% skim milk (BD, Franklin Lakes, NJ, USA) in Tris-buffered saline (TBS)–Tween 20 (0.1%) at room temperature for 1 h and then incubated with polyclonal goat anti-ORP5 antibody (Imgenex, San Diego, CA, USA), β-actin antibody (Cell Signaling Technology, Beverly, MA, USA), or V5 antibody (Invitrogen) for 1 h at room temperature.

Techniques: Expressing, Binding Assay, Transfection, Small Interfering RNA, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Polymerase Chain Reaction

Journal: Cancer science

Article Title: Oxysterol binding protein-related protein-5 is related to invasion and poor prognosis in pancreatic cancer.

doi: 10.1111/j.1349-7006.2008.00987.x

Figure Lengend Snippet: Fig. 4. Immunohistochemical staining of oxysterol binding protein-related protein (ORP)-5. The exp- ression level of ORP5 in pancreatic cancer specimens was analyzed in comparison with that in the acinar cells of the pancreas. (a) ORP5-negative pancreatic cancer tissue. (b) ORP5-positive pancreatic cancer tissue. ORP5 was highly expressed in the cytoplasm of cancer cells. (c) Weak expression of ORP5 in the acinar cells of the pancreas. Scale bar = 100 μm.

Article Snippet: The membrane was blocked with 5% skim milk (BD, Franklin Lakes, NJ, USA) in Tris-buffered saline (TBS)–Tween 20 (0.1%) at room temperature for 1 h and then incubated with polyclonal goat anti-ORP5 antibody (Imgenex, San Diego, CA, USA), β-actin antibody (Cell Signaling Technology, Beverly, MA, USA), or V5 antibody (Invitrogen) for 1 h at room temperature.

Techniques: Immunohistochemical staining, Staining, Binding Assay, Comparison, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Oxysterol-binding protein–related protein 5 (ORP5) promotes cell proliferation by activation of mTORC1 signaling

doi: 10.1074/jbc.RA117.001558

Figure Lengend Snippet: ORP5 depletion down-regulates mTORC1 signaling. A, HeLa/Mock and HeLa/ORP5 cells were treated with control or ORP5 siRNAs. Cells were starved in serum-free medium overnight prior to harvest for immunoblotting analysis. B, densitometry of activated p70 S6 kinase (p-S6K) in A (mean ± S.D.; *, p < 0.05; n = 4). C, densitometry of activated S6 ribosomal protein (p-S6) in A (mean ± S.D.; *, p < 0.05; **, p < 0.01; n = 4). D, HeLa/ORP5 cells were treated with control and two different ORP5 siRNAs. Cells were starved in serum-free medium overnight prior to harvest for immunoblotting analysis. E, densitometry of p-S6 in D (mean ± S.D.; *, p < 0.05; **, p < 0.01; n = 3). F, protein synthesis assay in HeLa/ORP5 cells treated with control or ORP5 siRNAs. Newly synthesized proteins in the cytosol were detected by the Click-iT® HPG Alexa Fluor® 488 protein synthesis assay kit. G, Click-iT HPG signals in F were quantified using ImageJ and plotted relative to the values of control siRNA-treated cells (mean ± S.D.; ****, p < 0.0001; n = 16–24 cells). H, PANC-1 cells were treated with control or ORP5 siRNAs for 72 h followed by harvest for immunoblotting analysis. I, PANC-1 cells were treated with control or ORP5 siRNAs for 72 h. Cells were imaged under a wide-field microscope. J, cell proliferation assay in PANC-1 cells treated with control siRNA or three different ORP5 siRNAs (mean ± S.D.; ****, p < 0.0001; n = 12).

Article Snippet: Antibodies used were rabbit polyclonal to mTOR (Cell Signaling Technology (CST), catalog no. 2983, clone 7C10); S6K (CST, catalog no. 9202); p-T389 S6K (CST, catalog no. 9205); S6 (CST, catalog no. 2217, clone 5G10); p-S240/244 S6 (CST, catalog no. 5364, clone D68F8); β-actin (CST, catalog no. 4970, clone 13E5); calnexin (CST, catalog no. 2433); GAPDH (CST, catalog no. 2118, clone 14C10); Akt (CST, catalog no. 9272); RFP (Abcam, ab167453); and ORP5 (Sigma, HPA038335); antibodies used were goat polyclonal to ORP5 (Abcam, ab59016); mouse monoclonal to LAMP1 (Santa Cruz Biotechnology, sc-20011, clone H4A3); RFP (Santa Cruz Biotechnology, sc-390909, clone E-8); actin (Abcam, ab8226); and GFP (Santa Cruz Biotechnology, sc-9996, clone B-2).

Techniques: Control, Western Blot, Synthesized, Microscopy, Proliferation Assay

Journal: The Journal of Biological Chemistry

Article Title: Oxysterol-binding protein–related protein 5 (ORP5) promotes cell proliferation by activation of mTORC1 signaling

doi: 10.1074/jbc.RA117.001558

Figure Lengend Snippet: ORP5 depletion down-regulates mTORC1 signaling. A, HeLa/Mock and HeLa/ORP5 cells were treated with control or ORP5 siRNAs. Cells were starved in serum-free medium overnight prior to harvest for immunoblotting analysis. B, densitometry of activated p70 S6 kinase (p-S6K) in A (mean ± S.D.; *, p < 0.05; n = 4). C, densitometry of activated S6 ribosomal protein (p-S6) in A (mean ± S.D.; *, p < 0.05; **, p < 0.01; n = 4). D, HeLa/ORP5 cells were treated with control and two different ORP5 siRNAs. Cells were starved in serum-free medium overnight prior to harvest for immunoblotting analysis. E, densitometry of p-S6 in D (mean ± S.D.; *, p < 0.05; **, p < 0.01; n = 3). F, protein synthesis assay in HeLa/ORP5 cells treated with control or ORP5 siRNAs. Newly synthesized proteins in the cytosol were detected by the Click-iT® HPG Alexa Fluor® 488 protein synthesis assay kit. G, Click-iT HPG signals in F were quantified using ImageJ and plotted relative to the values of control siRNA-treated cells (mean ± S.D.; ****, p < 0.0001; n = 16–24 cells). H, PANC-1 cells were treated with control or ORP5 siRNAs for 72 h followed by harvest for immunoblotting analysis. I, PANC-1 cells were treated with control or ORP5 siRNAs for 72 h. Cells were imaged under a wide-field microscope. J, cell proliferation assay in PANC-1 cells treated with control siRNA or three different ORP5 siRNAs (mean ± S.D.; ****, p < 0.0001; n = 12).

Article Snippet: Antibodies used were rabbit polyclonal to mTOR (Cell Signaling Technology (CST), catalog no. 2983, clone 7C10); S6K (CST, catalog no. 9202); p-T389 S6K (CST, catalog no. 9205); S6 (CST, catalog no. 2217, clone 5G10); p-S240/244 S6 (CST, catalog no. 5364, clone D68F8); β-actin (CST, catalog no. 4970, clone 13E5); calnexin (CST, catalog no. 2433); GAPDH (CST, catalog no. 2118, clone 14C10); Akt (CST, catalog no. 9272); RFP (Abcam, ab167453); and ORP5 (Sigma, HPA038335); antibodies used were goat polyclonal to ORP5 (Abcam, ab59016); mouse monoclonal to LAMP1 (Santa Cruz Biotechnology, sc-20011, clone H4A3); RFP (Santa Cruz Biotechnology, sc-390909, clone E-8); actin (Abcam, ab8226); and GFP (Santa Cruz Biotechnology, sc-9996, clone B-2).

Techniques: Control, Western Blot, Synthesized, Microscopy, Proliferation Assay