Journal: Scientific Reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: mTOR activity is required for optimal viral-protein synthesis. ( a ) Schematic of experimental protocol. ( b ) HeLa cells were transfected with pNL4-3, left untreated or treated with 250 nM Torin1 in medium containing AHA. At the indicated times, cell lysates were collected and Click-it chemistry was performed followed by immunoprecipitation with rabbit anti-p24 antibody. Membranes were probed with the indicated antibodies. ( c ) Densitometry quantification of de novo synthesized, biotinylated Gag from panel b was determined by ImageJ analysis. For each time point, the values presented in the graph are normalized against the total amount of Gag in the cell lysate (refer to Supplementary Fig. for total protein synthesis). Full-length blots are shown in Supplementary Fig. . ( d ) HeLa cells were transfected with either pcDNA3.1 or pNL4-3 and infected with L. major (lmj) WT or GP63 KO. Cell lysates were subjected to SDS-PAGE, immunoblotted and probed with the indicated antibodies against host (left panels) and viral (right panels) antigens.

Article Snippet: HIV-1-expressing cells were identified by viral RNA (vRNA) using fluorescence in situ hybridization (FISH) and were stained with antibodies against the lysosomal population of mTOR (Cell Signaling 7C10) and LAMP-1.

Techniques: Activity Assay, Transfection, Immunoprecipitation, Synthesized, Infection, SDS Page

Journal: Scientific Reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: HIV-1 does not maintain mTORC1 activation during nutrient deprivation. ( a ) HeLa cells were transfected with either pcDNA3.1 or pNL4-3 and starved for amino acids (aa) (and serum) (1 h) and subsequently recovered with complete media (serum free) for an additional 30 minutes. HIV-1-expressing cells were identified by viral RNA (vRNA) using fluorescence in situ hybridization (FISH) and were stained with antibodies against the lysosomal population of mTOR (Cell Signaling 7C10) and LAMP-1. Light-blue arrowheads identify HIV-1-expressing cells, while HIV-1-negative cells in the same field are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μm. ( b ) The percentage of mock or HIV-1-transfected cells showing perinuclear clustering of mTOR/LAMP-1 from panel a. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05). ( c ) HeLa cells were transfected with either pcDNA3.1 or pNL4-3 and starved for amino acids (aa) (1 h) and subsequently recovered with complete media for an additional 30 minutes. Cell lysates were subjected to SDS-PAGE, immunoblotted and probed with the indicated antibodies. Full-length blots are shown in Supplementary Fig. . ( d ) The graphs show the ratio of S6K1-pT389/total S6K1 for mock or HIV-1-transfected cells as in panel c. Values were normalized against untreated cells transfected with pcDNA3.1. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05); n.s.: no signifcant difference between the means.

Article Snippet: HIV-1-expressing cells were identified by viral RNA (vRNA) using fluorescence in situ hybridization (FISH) and were stained with antibodies against the lysosomal population of mTOR (Cell Signaling 7C10) and LAMP-1.

Techniques: Activation Assay, Transfection, Expressing, Fluorescence, In Situ Hybridization, Staining, SDS Page

Journal: Scientific Reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: HIV-1 redistributes LEL. ( a ) HeLa cells were transfected with either pcDNA3.1 (not shown) or pNL4-3 and left untreated of stressed with 500 µM Ars. HIV-1-expressing cells were identified using fluorescence in situ hybridization (FISH) to localize vRNA and stained using antibodies against mTOR (Cell Signaling 7C10) and LAMP-1. Light blue arrowheads identify HIV-1-expressing cells, while HIV-1-negative cells in the same field are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μ m. ( b ) The percentage of mock or HIV-1-transfected cells showing perinuclear clustering of mTOR/LAMP-1 from panel a. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05); n.s.: no signifcant difference between the means. ( c ) The distance of lysosomes from the nucleus was quantified from at least 60 cells using the Spots Tool in Imaris software. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05). ( d ) HeLa cells were co-transfected with either pcDNA3.1 or pNL4-3 and mRFP-ORP1L. HIV-1-expressing cells were identified using FISH to localize vRNA and stained for LAMP-1. Scale bar is 2 μm. ( e ) Diagram of the quantification analysis of the immunofluorescence intensity of LAMP-1 in concentric rings from the nucleus to the plasma membrane. Representative image from a pNL4-3 and mRFP-ORP1L transfected cell. ( f ) Quantitation of LAMP-1 immunofluorescence intensity in concentric rings spanning out from the nucleus in cells overexpressing mRFP-ORP1L co-transfected with either pcDNA3.1 or pNL4-3 (n = 40). Statistical significance was tested using a two-way ANOVA and P values are indicated by *(p < 0.05), **(p < 0.01) and ***(p < 0.001). ( g ) HIV-1-expression was induced by doxycycline (2 μg/mL) in an inducible THP-1-derived macrophage cell line for 72 h. Cells were left untreated or stressed with Ars for 1 h before fixation. HIV-1-expressing cells were identified by Gag-GFP expression and stained for mTOR (Cell Signaling 7C10) and LAMP-1.

Article Snippet: HIV-1-expressing cells were identified by viral RNA (vRNA) using fluorescence in situ hybridization (FISH) and were stained with antibodies against the lysosomal population of mTOR (Cell Signaling 7C10) and LAMP-1.

Techniques: Transfection, Expressing, Fluorescence, In Situ Hybridization, Staining, Software, Immunofluorescence, Clinical Proteomics, Membrane, Quantitation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: RagA and RagB are necessary for HIV-1 to control LEL trafficking and for the viral budding and release. ( a ) HeLa cells, treated with non-silencing (NS) siRNA or siRNAs targeting RagA and RagB mRNAs, were transfected with empty vector control, pCDNA3.1 or HIV-1 using pNL4-3/GagmRFP, and then mock treated or treated with Ars. Cells were stained for mTOR (Cell Signaling 7C10) and LAMP-1. Light blue arrowheads identify HIV-1-expressing cells, while HIV-1-negative cells are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μ m. ( b ) The graphs represent the fluorescence intensities of mTOR (green) and LAMP-1 (blue) across the dashed lines from panel a. ( c ) The percentage of mock or HIV-1-transfected cells showing perinuclear clustering of LEL from panel a. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05); n.s.: no signifcant difference between the means. ( d ) HeLa cells were co-transfected with the HIV-1 pNL4-3 and either non-sense (NS) siRNA or siRNA against RagA and RagB. HIV-1 particles were collected from the supernatant after filtration and ultracentrifugation. Cell lysates and virus particles were separated by SDS-PAGE, transferred to nitrocellulose and probed with the indicated antibodies. siRNA-mediated fold changes in RagA, RagB and virus production are indicated below insets in panel d in this representative experiment. Full-length blots are shown in Supplementary Fig. . ( e ) HeLa cells were co-transfected with the HIV-1 pNL4-3 and either non-sense (NS) siRNA or siRNA against RagA and RagB. Cells were fixed 24 h after transfection and stained for p24. ( f ) The percentage of HIV-1 and siRagA/B transfected cells showing peripheral accumulation of Gag from e. The results are presented as the mean ± S.D. from three different experiments. ( g ) HeLa cells were transfected as in e and subjected to transmission electron microscopy.

Article Snippet: HIV-1-expressing cells were identified by viral RNA (vRNA) using fluorescence in situ hybridization (FISH) and were stained with antibodies against the lysosomal population of mTOR (Cell Signaling 7C10) and LAMP-1.

Techniques: Control, Transfection, Plasmid Preparation, Staining, Expressing, Fluorescence, Filtration, Virus, SDS Page, Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases

doi: 10.1038/s41598-017-05410-0

Figure Lengend Snippet: RagA and RagB interact with Gag and Vif. ( a ) Lysates from control pcDNA3.1- or pNL4-3-transfected HeLa cells were subjected to immunoprecipitation with RagA antibodies. Samples were subjected to SDS-PAGE followed by transfer to nitrocellulose membranes and probed with the indicated antibodies. Full-length blots are shown in Supplementary Fig. . ( b ) Lysate from pcDNA3.1-, pNL4-3-, pNLXX−, pNLXX+ pSVGag− and pNLVif–transfected HeLa cells were subjected to immunoprecipitation with RagA and RagB antibodies or isotype rabbit IgG control. The blots were probed with anti-p24, anti-Vif, anti-RagA and anti-RagB antibodies. Full-length blots are shown in Supplementary Fig. . ( c ) HeLa cells were transfected with either pNLXX or pNLVif- and left untreated (Unt) or stressed with 500 µM Ars for 1 h. HIV-1-expressing cells were identified using FISH to localize vRNA and stained using antibodies against mTOR (Cell Signaling) and LAMP-1. Light blue arrowheads identify pNLXX or pNLVif- expressing cells, while pNLXX or pNLVif- negative cells in the same field are indicated with green arrowheads. A dashed line contours individual cells. Scale bars are 10 μm. ( d ) The percentage of mock, pNLXX or pNLVif- transfected cells showing perinuclear clustering of mTOR/LAMP-1 from panel c. The results are presented as the mean ± S.D. from three different experiments. P value is indicated by *(p < 0.05).

Article Snippet: HIV-1-expressing cells were identified by viral RNA (vRNA) using fluorescence in situ hybridization (FISH) and were stained with antibodies against the lysosomal population of mTOR (Cell Signaling 7C10) and LAMP-1.

Techniques: Control, Transfection, Immunoprecipitation, SDS Page, Expressing, Staining

Journal: Scientific Reports

Article Title: KIN17 facilitates the initiation and progression of renal tumor progression through the PI3K-AKT-mTOR pathway

doi: 10.1038/s41598-026-35851-5

Figure Lengend Snippet: KIN17 positively regulates PI3K/AKT/mTOR expression in RCC and PF-04691502 can reverse the pathway protein expression and biological function. ( A ) Predict effects of KIN17 knockdown on the pathways of RCC cells (ACHN and 786–0) in vitro by kegg and GSEA. ( B ) p-mTOR, mTOR, p-AKT, AKT, p-PI3K and PI3K protein levels were measured by WB using the paired RCC cells (ACHN and 786–0) group and knockdown KIN17 group. ( C ) p-mTOR, mTOR, p-AKT, AKT, p-PI3K and PI3K protein levels were measured by WB using the paired RCC vector cells group (VE groups), overexpression KIN17 group (OE groups), VE groups + PF-04691502 and OE groups + PF-04691502. ( D ) N-cadherin and Vimentin protein levels were measured by WB using the paired RCC cells (ACHN and 786–0) in knockdown KIN17 group and different using PF-04691502 groups.

Article Snippet: The membranes were then blocked with 5% non-fat milk in TBS/Tween (0.05% Tween-20 in TBS) for 2 h. Membranes were incubated with primary antibodies including KIN17(Santa Cruz, Catalog number: sc-32768, 1:500), AKT (Proteintech, Catalog number: 60203–2-Ig, 1:10,000), mTOR (Proteintech, Catalog number: 66888–1-Ig, 1:10,000), PI3K(CST, Catalog number: 11889, 1:1000), p-AKT(CST, Catalog number: 4060S, 1:2000), p-mTOR(CST, Catalog number: 5536S, 1:1000), p-PI3K(CST, Catalog number: 17366S, 1:1000), N-cadherin (Proteintech, Catalog number: 22018–1-AP, 1:10,000), Vimentin (Proteintech, Catalog number: 10366–1-AP, 1:50,000) and GAPDH (Proteintech, 60,004–1-lg, 1:10,000) overnight at 4°C and then with secondary antibodies (anti-rabbit or anti- mouse; Proteintech) for room temperature.

Techniques: Expressing, Knockdown, In Vitro, Plasmid Preparation, Over Expression

Journal: Scientific Reports

Article Title: KIN17 facilitates the initiation and progression of renal tumor progression through the PI3K-AKT-mTOR pathway

doi: 10.1038/s41598-026-35851-5

Figure Lengend Snippet: KIN17 promotes tumor growth and suppression of tumor growth by PF-04691502 in vivo ( A ) Representative images of xenograft tumors 786–0 cells with expression of shNC and shKIN17 (n = 7 in each group). Tumor volume and tumor weight were analyzed. ( B ) Representative H&E staining and immunohistochemstry staining of Ki67, TUNEL, KIN17, p-mTOR and p-AKT in the xenograft tumor tissues (× 400 magnification). ( C ) Representative images of xenograft tumors 786–0 cells with expression of Vector, Vector + PF-04691502, OE and OE + PF-04691502 (n = 8 in each group). Tumor volume and tumor weight were analyzed. ( D ) Representative H&E staining and immunohistochemstry staining of Ki67, TUNEL, KIN17, p-mTOR and p-AKT in the xenograft tumor tissues (× 400 magnification). The error bars represent the mean ± SD of triplicate technical replicates. * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: The membranes were then blocked with 5% non-fat milk in TBS/Tween (0.05% Tween-20 in TBS) for 2 h. Membranes were incubated with primary antibodies including KIN17(Santa Cruz, Catalog number: sc-32768, 1:500), AKT (Proteintech, Catalog number: 60203–2-Ig, 1:10,000), mTOR (Proteintech, Catalog number: 66888–1-Ig, 1:10,000), PI3K(CST, Catalog number: 11889, 1:1000), p-AKT(CST, Catalog number: 4060S, 1:2000), p-mTOR(CST, Catalog number: 5536S, 1:1000), p-PI3K(CST, Catalog number: 17366S, 1:1000), N-cadherin (Proteintech, Catalog number: 22018–1-AP, 1:10,000), Vimentin (Proteintech, Catalog number: 10366–1-AP, 1:50,000) and GAPDH (Proteintech, 60,004–1-lg, 1:10,000) overnight at 4°C and then with secondary antibodies (anti-rabbit or anti- mouse; Proteintech) for room temperature.

Techniques: In Vivo, Expressing, Staining, TUNEL Assay, Plasmid Preparation

Journal: Artificial cells, nanomedicine, and biotechnology

Article Title: Carvacrol nanoemulsion evokes cell cycle arrest, apoptosis induction and autophagy inhibition in doxorubicin resistant-A549 cell line.

doi: 10.1080/21691401.2018.1434187

Figure Lengend Snippet: Figure 5. CANE abolishes autophagy in A549DR. (A) CANE was treated to A549DR cells for 24 h and autophagic protein levels detected such as LC-3, p62, Beclin-1, mTOR, ATG5 and ATG7. Densitometry analysis of the respective proteins was evaluated by Image J software, and results were normalized with b-actin with respect to controls. (B) For further confirmation, autophagic vacoule formation detected with the help of fluorogenic dye LysoTracker V R Red, DAPI was used as counter stain to stain nucleus. Acridine orange was used to detect the autopaghic vacoule formation. Image J software was used for quantification of oxidative stress. Images were taken at 40 magnification [scale bar¼ 0.1 mm]. Each value in the graph represents as the mean ± SD of three independent experiments. Values with different superscripts differ significantly from each other (p < .05).

Article Snippet: Carvacrol, 3-Methyladenine (3-MA), N-acetyl-L-cysteine (NAC), primary antibodies against b-actin, JNK, p-JNK, caspase-3, caspase-9, Bax, Bcl2, cytochrome C, CDK2, CDK4, CDK6, Cyclin E, Cyclin D1, p21, LC-3, ATG5, ATG7, mTOR, p62, Beclin-1 as well as horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Software, Staining