Journal: Cells

Article Title: Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

doi: 10.3390/cells13131141

Figure Lengend Snippet: Figure 3. Flow cytometric determination of the level of XBP1 (mNeonGreen) protein in heat-stressed U2OS cells. Cells were heat-stressed at 40 ◦C or 42 ◦C for one hour, followed by six hours of recovery at 37 ◦C. The Kolmogorov–Smirnov test was performed to analyze the equality of distributions. Samples treated at 40 or 42 ◦C differed from the control (37 ◦C) significantly (p < 0.05).

Article Snippet: To construct the U2OS cell line expressing fluorescently labeled XBP1 (mNeonGreen), 200 ng linearized pLHCX-XBP1 mNeonGreen plasmid NLS (Addgene plasmid #115971; http://n2t.net/addgene:115971; RRID: Addgene_115971), a gift from David Andrews [23], was transfected into 106 U2OS cells using the Amaxa Nucleofector System with the Cell Line Nucleofector Kit V (catalog no: VCA-1003, Lonza, Cologne, Germany) utilizing program X-001 according to the manufacturer’s protocol.

Techniques: Control

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Movement of free cholesterol in MA-10 cells. A, confocal microscope images of cells transfected with mCherry-D4. Images a–e, time course of one control (untreated) cell; images f–j, time course of one cell treated with Bt2cAMP (dbcAMP). Scale bar, 10 μm. B, time course of fluorescence intensity at the plasma membrane of control and Bt2cAMP-treated cells transfected with mCherry-D4. C, time course of progesterone levels in untransfected cell and cells transfected with mCherry-D4 after treatment with Bt2cAMP. D, confocal microscope images of cells transfected with mCherry-D4 before treatment and 30 min after treatment with methyl-β-cyclodextrin. Scale bar, 10 μm. E, confocal microscope images of cells before treatment and cells incubated with U18666A for 4 h and then treated with Bt2cAMP for 2 h. Scale bar, 10 μm. F, progesterone production in mCherry-D4-transfected cells treated with U18666A, U18666A + Bt2cAMP, Bt2cAMP and control (untreated). Data represent means ± S.D. of at least three independent experiments performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (***) or t test (###) were used to calculate statistical significance; ***, ###, p < 0.001; Scale bars, 10 μm; dbcAMP, dibutyryl cAMP.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Microscopy, Transfection, Fluorescence, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Intracellular cholesterol distribution in MA-10 cells expressing mCherry-D4. A and B, confocal images of control and mCherry-D4-expressing MA-10 cells fixed with 4% paraformaldehyde and stained with 50 μg/ml filipin. Scale bars, 10 μm. C and D, 1 mm Bt2cAMP (dbcAMP)-treated MA-10 cells with and without (w/o) mCherry-D4 expression, fixed, and stained with 50 μg/ml filipin. Scale bars, 10 μm. E, fluorescence intensity of filipin staining at the plasma membrane in control and Bt2cAMP-treated MA-10 cells with or without mCherry-D4 expression. Data represent mean ± S.D. of at least 12 different cells performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (*) were used to calculate statistical significance. ***, p < 0.001. F, protein content in whole cell homogenates (WH), cytoplasm (C), endosomes (Endo), mitochondria (Mito), and plasma membrane (PM) isolates were quantified using Bradford assay, and 15 μg of protein were separated in a Novex NuPAGE BisTris 4–12% (w/v) precast gel (Invitrogen) and transferred to PVDF membranes for standard Western blotting. Proteins were detected with anti-Rab5 (1:2000, Abcam), anti-VDAC1 (1:5000, Abcam), anti-PMCA1 (1:1000, Abcam), and anti-β-actin (1:1000, Abcam), followed by appropriate secondary HRP-conjugated antibodies (1:1000, Cell Signaling Technology). G, organelle cholesterol distribution was measured by LC-MS/MS in whole cell homogenates, cytoplasm, endosomes, mitochondria, and plasma membrane isolates from control and mCherry-D4-expressing MA-10 cells.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Expressing, Staining, Fluorescence, Bradford Assay, Western Blot, Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Effect of inhibitors of the proteins associated with the steroidogenic metabolon on cholesterol trafficking from the plasma membrane in MA-10 cells transfected with mCherry-D4. A–D, time course of mCherry-D4 fluorescence associated with the plasma membrane in the presence of various inhibitors in the presence or absence of Bt2cAMP. A, aminoglutethimide (AMG), an inhibitor of CYP11A1. B, erastin, an inhibitor of VDAC. C, two concentrations of the START domain ligand, an inhibitor of the STAR protein. D, two concentrations of the CRAC domain ligand, an inhibitor of TSPO. E, progesterone production 2 h after exposure of the cells to various inhibitors. Data represent means ± S.D. of at least three independent experiments performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (*) was used to calculate statistical significance; *, p < 0.05; **, ##, p < 0.01; ***, p < 0.001. CRAC, cholesterol recognition amino acid consensus sequence; CYP11A1, cytochrome P450 side chain cleavage enzyme; STAR, steroidogenic acute regulatory protein; TSPO, translocator protein; VDAC, voltage-dependent anion channel.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Transfection, Fluorescence, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Effect of steroids on trafficking of free cholesterol in mCherry-D4-expressing MA-10 cells. A and B, mCherry-D4 fluorescence intensity associated with the plasma membrane in the presence or absence of Bt2cAMP (dbcAMP). A, cells treated with the steroid intermediate 22R-HC. B, cells treated with the stereoisomer 22S-HC. C and D, progesterone levels in cells 2 h after treatment with 22S-HC or 22R-HC: either C, without Bt2cAMP, or D, with Bt2cAMP. E and F, mCherry-D4 fluorescence intensity associated with the plasma membrane in cells treated with or without Bt2cAMP and either E, progesterone, or F, pregnenolone. Data represent means ± S.D. of at least three independent experiments performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (*) or t test (#) were used to calculate statistical significance; *, #, p < 0.05; **, ##, p < 0.01; ***, ###, p < 0.001. 22R-HC, 22(R)-hydroxycholesterol; 22S-HC, 22(S)-hydroxycholesterol; dbcAMP, dibutyryl cAMP.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Identification of organelles involved in cholesterol trafficking during steroid biosynthesis in mCherry-D4-expressing MA-10 cells. Co-localization of mCherry-D4 with organelles was assessed with appropriate fluorescent markers in living cells. A, images of the fluorescence staining results. 1st column, untreated cells; 2nd column, Bt2cAMP (dbcAMP)-treated cells. Scale bar, 10 μm. B, correlation coefficients between images and organelles based on quantification of co-localization of images with mCherry-D4, using ImageJ with the plug-in co-localization using Pearson's correlation coefficient. Data represent means ± S.D. (n = 9 cells per group). *, p < 0.05. dbcAMP, dibutyryl cAMP. C, 72-h post-transfection with siRNA targeting NPC-2 resulted in reduced levels of NPC-2 levels, confirmed by immunoblotting with anti-NPC-2 (1:1000, Santa Cruz Biotechnology), and GAPDH was used as control using anti-GAPDH (1:1000, Trevigen). D and E, to assess the role NPC-2 in acute steroidogenesis, NPC-2 knocked down MA-10 cells were treated with 1 mm Bt2cAMP for 2 h and progesterone production was measured by RIA before and after stimulation. Statistical analysis was performed using Student's t test. *, p < 0.05.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Expressing, Fluorescence, Staining, Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: No change in the plasma membrane outer leaflet cholesterol of mCherry-D4-expressing MA-10 cells with and without Bt2cAMP treatment. A and B, cells were fixed with paraformaldehyde and treated with recombinant EGFP-D4 to assess binding to the outer leaflet of the plasma membrane. Where a decrease in the inner leaflet binding mCherry-D4 was noticed, no change in the binding of recombinant EGFP-D4 was observed. Scale bar, 10 μm.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Expressing, Recombinant, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Movement of free cholesterol in mCherry-D4-transfected R2C cells, which constitutively synthesize steroids. A, confocal microscope images of cells with and without treatment with Bt2cAMP. Scale bar, 10 μm. B, time course of mCherry-D4 fluorescence intensity associated with the plasma membrane. C, progesterone production measured in control and mCherry-D4-transfected cells, before and after 2 h of Bt2cAMP treatment. Data represent means ± S.D. of at least three independent experiments performed in triplicate. dbcAMP, dibutyryl cAMP.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Transfection, Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells *

doi: 10.1074/jbc.M116.740928

Figure Lengend Snippet: Cholesterol trafficking analysis in primary rat Leydig cells infected with lentivirus containing mCherry-D4. A, confocal microscope images of cells with and without addition of Bt2cAMP. Scale bar, 10 μm. B, time course of mCherry-D4 fluorescence intensity at the plasma membrane of control and Bt2cAMP-treated cells. C, testosterone content of control cells and cells infected with lentivirus before and after a 2-h treatment with Bt2cAMP. Testosterone was measured just after the cells were isolated (0 h), in control (uninfected) cells 18 h after incubation and 18 h after infection. Data represent means ± S.D. of at least three independent experiments performed in triplicate; two-way ANOVA followed by Bonferroni's post hoc test (*) was used to calculate statistical significance. *, p < 0.05; **, p < 0.01; ***, p < 0.001. dbcAMP, dibutyryl cAMP.

Article Snippet: The D4 cDNA coding sequence was subcloned into the HIV-based lentiviral expression vector, pLVX-mCherry-C1 (Clontech), at the EcoRI and BamHI sites using the following primers: 5′-gaattcgtacaagAAGCTTaaggg-3′ and 5′-ggatccGCGGGTTTAAACCTCGAG-3′.

Techniques: Infection, Microscopy, Fluorescence, Isolation, Incubation

Journal: Cell reports

Article Title: A Hyperactive Signalosome in Acute Myeloid Leukemia Drives Addiction to a Tumor-Specific Hsp90 Species

doi: 10.1016/j.celrep.2015.10.073

Figure Lengend Snippet: (A and B) Immunoblot (A) and flow cytometry (B) of FL5.12 cells after 24 hr treatment with JAKi, AKTi, or PU-H71 (PU) in the presence or absence of IL-3. (C) Percent dead cells in FL5.12 cells after 48 hr treatment with JAKi, AKTi, or PU. (D and E) Immunoblots for pAKT (D) and percent apoptotic cells (E) of vector control- or mAKT-transfected cells treated with PU-H71, AKTi ± doxycycline (DOX) for 24 hr or 48 hr, respectively. (F) Percentage of mCherry+ cells KG-1/N51 or KG-1/WT over 30-day cultures. (G) Percentage of apoptotic cells after 48 hr treatment with PU-H71. (H and I) Phosphorylated protein levels in cells evaluated by immunoblot (H) or flow cytometry (I) (fold change of MFI for KG-1/N51 relative to KG-1/WT). (J) Immunoblots of Hsp90-interacting proteins as isolated by PU-beads. (K) Immunoblots for Hsp90 and FLT3 immunoprecipitated with an anti-FLT3 antibody. (L) Correlation of the apoptotic sensitivity to PU-H71 (x axis) and to AKTi, JAKi, MEK1/2i, or p38i (y axis) in cells treated for 48 hr. (M) Percent cell death for FLT3-ITD− or FLT3-ITD+ primary AML cells after 48 hr exposure to PU-H71. (N) Hsp90 expression of cells from (M) as measured by flow cytometry. Each symbol represents an individual sample. Values denote mean ± SEM. *p < 0.05; **p < 0.01;***p < 0.001. See also .

Article Snippet: Wild type FLT3 (WT-FLT3) and FLT3-ITD mutant (N51-FLT3) cDNA were PCR amplified from MSCV-WTFLT3-IRES-GFP and MSCV-N51FLT3-IRES-GFP (kindly provided by Dr. Craig Jordan) respectively. pLVX-WTFLT3-mCherry and pLVX-N51FLT3-mCherry plasmids were generated by subcloning WT-FLT3 and N51-FLT3 cDNA to the SpeI/BamHI restriction sites of pLVX-EF1-IRES-mCherry vector (Clontech).

Techniques: Western Blot, Flow Cytometry, Plasmid Preparation, Transfection, Isolation, Immunoprecipitation, Expressing