Journal: Oncology Reports

Article Title: Combined inhibition of ACLY and CDK4/6 reduces cancer cell growth and invasion

doi: 10.3892/or.2022.8469

Figure Lengend Snippet: Palb activation of ACLY is dependent on AKT. (A) MDA-MB-231 and Panc1 cells were treated with 0.1% DMSO or 1 µM P for 96 h. Cellular protein was quantified using Bradford assays and equal amounts of protein were separated by SDS-PAGE. (B) MDA-MB-231 or Panc1 cells were treated with 0.1% DMSO and NT RNA as a control. Cells treated with 1 µM Palb for 96 h were also subjected to AKT knockdown. Protein analysis, western blotting and antibodies utilized was as described in the Materials and methods. Results shown were repeated twice, and Palb, palbociclib; NT, non-targeting; ACLY, ATP citrate lyase; Rb, retinoblastoma; RNAi, RNA interference; p, phosphorylated.

Article Snippet: MDA-MB-231 or Panc1 cells were transfected with AKT small interfering RNA (cat. no. 6211; Cell Signaling Technology, Inc.) or NT RNA as a control (cat. no. 6568; Cell Signaling Technology, Inc.) at 100 nM at 37°C for 48 h prior to cell lysis, using Lipofectamine RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Activation Assay, SDS Page, Western Blot

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: List of reagents and antibodies.

Article Snippet: 41 , PLK1 siRNA, , 100 nM final conc , 6292, AM5133 , Cell Signaling Technology, Thermo Fisher Scientific.

Techniques: Concentration Assay, SYBR Green Assay, Protease Inhibitor, Plasmid Preparation, Magnetic Beads

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: Bar graphs showing mitosis scores in the Emory ( A ) and Dekalb ( B ) cohorts. C Heatmap showing the expression levels of various kinases in the TCGA BC dataset. D , E Bar graphs showing the expression levels of PLK1 ( D ) and AURKB ( E ) in AA ( n = 3) and EA ( n = 3) TNBC cell lines. F – H Representative IHC images of PLK1 and AURKB ( F ) and quantification bar graphs showing PLK1 ( G ) and AURKB ( H ) levels in grade- and stage-matched AA and EA patients with TNBC (Dekalb cohort). I Immunoblot showing PLK1 and AURKB protein levels in AA and EA TNBC cell lines ( n = 3 each). FPKM fragments per kilobase of transcript per million mapped reads. Bars indicate mean ± SEM. Unpaired two-tailed Student’s t -test with Welch’s correction was used to determine statistical significance (* P < 0.05, *** P < 0.0005, ns = non-significant). The scale bar represents 100 µm.

Article Snippet: 41 , PLK1 siRNA, , 100 nM final conc , 6292, AM5133 , Cell Signaling Technology, Thermo Fisher Scientific.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: Expression of various kinases in AA and EA patients with TNBC (TCGA dataset).

Article Snippet: 41 , PLK1 siRNA, , 100 nM final conc , 6292, AM5133 , Cell Signaling Technology, Thermo Fisher Scientific.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: A , B Immunoblots showing the levels of PLK1, survivin, p-survivin (S20), and β-actin after PLK1 silencing ( A , B ) or inhibition ( C ) in AA and EA TNBC cell lines. D – F Immunoblots showing the levels of AURKB, survivin, p-survivin (T117), and β-actin after AURKB silencing ( D , E ), or inhibition ( F ) in AA and EA TNBC cell lines.

Article Snippet: 41 , PLK1 siRNA, , 100 nM final conc , 6292, AM5133 , Cell Signaling Technology, Thermo Fisher Scientific.

Techniques: Western Blot, Inhibition

Journal: Cell Death & Disease

Article Title: PLK1 and AURKB phosphorylate survivin differentially to affect proliferation in racially distinct triple-negative breast cancer

doi: 10.1038/s41419-022-05539-5

Figure Lengend Snippet: A – F Representative IHC images ( A , B ) and bar graphs ( C – F ) showing Ki-67 and survivin levels in AA ( C , E ) and EA ( D , F ) TNBC xenografts under various treatment conditions. G , H Immunoblots showing the levels of p-survivin (T117), p-survivin (S20), total survivin, AURKB, PLK1, and β-actin in AA ( G ) and EA ( H ) fresh-frozen xenograft tumor lysates from mice treated with volasertib, barasertib, or their combination ( n = 12 per treatment group). Bars represent mean ± SEM. Unpaired two-tailed Student’s t -test with Welch’s correction was used to determine statistical significance (**** P < 0.00005, ns = non-significant). The scale bar represents 100 µm.

Article Snippet: 41 , PLK1 siRNA, , 100 nM final conc , 6292, AM5133 , Cell Signaling Technology, Thermo Fisher Scientific.

Techniques: Western Blot, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Activation of ERK1/2 signaling was necessary for venlafaxine-induced apoptosis. (A) Representative western-blot bands and quantification of the MAPK signal molecules abundances in MV3 cells treated with venlafaxine (10 μM) for 0–8 h (B) MV3 cells were treated with vehicle, JNK1/2 inhibitor SP600125 and MERK/ERK inhibitor PD98059 for 72 h. Cell viability were measured by CCK-8 kits at 72 h (C) MV3 cells were transfected with siRNA control, JNK1/2 siRNA, and ERK siRNA for 72 h. Cell viability were measured by CCK-8 kits at 72 h (D,E) Representative western-blot bands and quantification of Nur77 abundances in MV3 cells treated with venlafaxine (10 μM) for 8 h, or transfected with siRNA control, JNK1/2 siRNA, and ERK siRNA for 20 h (F) MV3 cells were immunostained with Bcl-2 and Nur77 antibodies and visualized by confocal microscopy. N = 3, *, p < .05, **, < .01, ***, p < .001 vs. control.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Activation Assay, Western Blot, CCK-8 Assay, Transfection, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Cellular viability was correlated to Nur77 expression induced by venlafaxine. (A) Chemical structure of venlafaxine. (B) Dose-dependent inhibition of venlafaxine on the growth inhibition of MV3 cells at 72 h. (C) Relative mRNA expressions of Nur77 in MV3 cells were quantified after treatment with venlafaxine (10 μM) for 0–8 h. (D) The protein expression of Nur77 in MV3 cells was detected by Western blot after treatment with venlafaxine (10 μM) for 0–8 h. Intensity of the protein bands was quantified and normalized to loading control GAPDH. N = 3, ***, p < .001 vs. control (0 h).

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Expressing, Inhibition, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Venlafaxine induced Nur77 mitochondrial targeting and ROS production in MV3 cells. (A) MV3 cells treated with venlafaxine (10 μM) for 0–8 h were immunostained with Bcl-2 and Nur77 antibodies and visualized by confocal microscopy. (B) MV3 cells treated with the cellular ROS assay kit and visualized by confocal microscopy.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Confocal Microscopy, ROS Assay

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Nur77 expression was necessary for venlafaxine-induced apoptosis. MV3 cells were transfected with siRNA control or Nur77 siRNA, following by treatment with venlafaxine (10 μM) for 0–8 h. (A) The protein expression of BAX-2, caspase 3 and cleaved PARP in MV3 cells was detected by western blot. Inte cleaved nsity of the protein bands was quantified and normalized to loading control GAPDH. (B) Apoptosis assays in MV3 cells were conducted by flow cytometry. (C) The apoptotic cells were detected by TUNEL assay. (D) MV3 cells treated with the cellular ROS assay kit and visualized by confocal microscopy. N = 3, **, p < .01, ***, p < .001 vs. control.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Expressing, Transfection, Western Blot, Flow Cytometry, TUNEL Assay, ROS Assay, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Activation of ERK1/2 signaling was necessary for venlafaxine-induced apoptosis. (A) Representative western-blot bands and quantification of the MAPK signal molecules abundances in MV3 cells treated with venlafaxine (10 μM) for 0–8 h (B) MV3 cells were treated with vehicle, JNK1/2 inhibitor SP600125 and MERK/ERK inhibitor PD98059 for 72 h. Cell viability were measured by CCK-8 kits at 72 h (C) MV3 cells were transfected with siRNA control, JNK1/2 siRNA, and ERK siRNA for 72 h. Cell viability were measured by CCK-8 kits at 72 h (D,E) Representative western-blot bands and quantification of Nur77 abundances in MV3 cells treated with venlafaxine (10 μM) for 8 h, or transfected with siRNA control, JNK1/2 siRNA, and ERK siRNA for 20 h (F) MV3 cells were immunostained with Bcl-2 and Nur77 antibodies and visualized by confocal microscopy. N = 3, *, p < .05, **, < .01, ***, p < .001 vs. control.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Activation Assay, Western Blot, CCK-8 Assay, Transfection, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Anti-cancer efficacy of venlafaxine in mice. BALB/c nude mice bearing MV3 or Nur77 KO MV3 xenograft tumors were treated with venlafaxine (20 mg/kg, i.p.) or its vehicle once daily from week 1 to week 5 after implantation of MV3 or Nur77 KO MV3 cells. (A) Tumor tissues were immunostained with Nur77 and JNK1/2 antibodies and visualized by confocal microscopy. (B) Venlafaxine reduced cancer cell growth in nude mice through Nur77. One of five similar experiments is shown.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Venlafaxine-induced apoptosis in vivo . BALB/c nude mice bearing WT MV3 or Nur77 KO MV3 xenograft tumors were treated with venlafaxine (20 mg/kg, i.p.) or its vehicle once daily from week 1 to week 5 after implantation of MV3 or Nur77 KO MV3 cells. (A) Representative western-blot bands and quantification of BAX-2, cleaved caspase 3 and cleaved PARP abundances in tumor tissues. N = 5, *, p < .05, **, < .01, ***, p < .001 vs. MV3 vehicle control. ### , p < .001 vs. MV3 venlafaxine control. (B) Tumor tissues were immunostained with cleaved caspase 3 and cleaved PARP antibodies and visualized by confocal microscopy. (C) Tumor tissues were immunostained with TUNEL assay kits and antibodies and Ki-67 visualized by confocal microscopy. One of five similar experiments is shown.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: In Vivo, Western Blot, Confocal Microscopy, TUNEL Assay

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Activation of ERK1/2 signaling was necessary for venlafaxine-induced apoptosis. (A) Representative western-blot bands and quantification of the MAPK signal molecules abundances in MV3 cells treated with venlafaxine (10 μM) for 0–8 h (B) MV3 cells were treated with vehicle, JNK1/2 inhibitor SP600125 and MERK/ERK inhibitor PD98059 for 72 h. Cell viability were measured by CCK-8 kits at 72 h (C) MV3 cells were transfected with siRNA control, JNK1/2 siRNA, and ERK siRNA for 72 h. Cell viability were measured by CCK-8 kits at 72 h (D,E) Representative western-blot bands and quantification of Nur77 abundances in MV3 cells treated with venlafaxine (10 μM) for 8 h, or transfected with siRNA control, JNK1/2 siRNA, and ERK siRNA for 20 h (F) MV3 cells were immunostained with Bcl-2 and Nur77 antibodies and visualized by confocal microscopy. N = 3, *, p < .05, **, < .01, ***, p < .001 vs. control.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Activation Assay, Western Blot, CCK-8 Assay, Transfection, Confocal Microscopy

Journal: Frontiers in Pharmacology

Article Title: Venlafaxine, an anti-depressant drug, induces apoptosis in MV3 human melanoma cells through JNK1/2-Nur77 signaling pathway

doi: 10.3389/fphar.2022.1080412

Figure Lengend Snippet: Anti-cancer efficacy of venlafaxine in mice. BALB/c nude mice bearing MV3 or Nur77 KO MV3 xenograft tumors were treated with venlafaxine (20 mg/kg, i.p.) or its vehicle once daily from week 1 to week 5 after implantation of MV3 or Nur77 KO MV3 cells. (A) Tumor tissues were immunostained with Nur77 and JNK1/2 antibodies and visualized by confocal microscopy. (B) Venlafaxine reduced cancer cell growth in nude mice through Nur77. One of five similar experiments is shown.

Article Snippet: MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h ( ).

Techniques: Confocal Microscopy

Journal: Virus Evolution

Article Title: Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

doi: 10.1093/ve/veac105

Figure Lengend Snippet: AGM-evolved SNPs and rhtrs1 duplication do not fully rescue virus replication in human-derived cells. (A) A549 cells (left bars) or A549 PKR −/− cells (right bars) were infected with the indicated viruses (MOI = 0.1). Two dpi, titers were determined by serial dilution on permissive BSC40 cells. Columns represent the mean of three independent biological replicates. Error bars indicate ± one SD. Differences between samples were determined by multiple unpaired t- tests. Samples with adjusted P -values < 0.05 (Holm–Šídák method) are indicated by brackets with P -values indicated above. (B) A549 cells were infected with the indicated viruses (MOI = 3.0). One dpi, cell lysates were analyzed by immunoblotting with the indicated antibodies. Data are representative of three independent biological replicates.

Article Snippet: Membranes were blocked with 5 per cent non-fat milk dissolved in TBST (20 M Tris, 150 mM NaCl, 0.1 per cent Tween 20, pH 7.4) for 1 h and probed with one of the following primary antibodies: anti-PKR (sc-6282; Santa Cruz Biotechnology, Inc.), anti-phospho-PKR (ab32036; Abcam), anti-eIF2α or anti-phospho-eIF2α (Ser51) antibody (9722 and 9721, respectively; Cell Signaling Technology), anti-TRS1 999 , or anti-actin (A2066; Sigma).

Techniques: Derivative Assay, Infection, Serial Dilution, Western Blot

Journal: Virus Evolution

Article Title: Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

doi: 10.1093/ve/veac105

Figure Lengend Snippet: RNase L mediates a second barrier to VACVΔEΔK + RhTRS1 replication in human cells. (A) A549 cells or A549 PKR −/− cells were infected with the indicated viruses (MOI = 3.0). One dpi, total RNA was harvested and visualized on an agarose gel + 1 per cent bleach. (B) A549 cells (left bars) or A549 RNase L −/− cells (right bars) were infected with the indicated viruses (MOI = 0.1). Two dpi, titers were determined by serial dilution on permissive BSC40 cells. Columns represent the mean of three independent biological replicates. Error bars indicate ± one SD. Differences between samples were determined by multiple unpaired t -tests. Samples with adjusted P -values < 0.05 (Holm–Šídák method) are indicated by brackets with P -values indicated above. (C) A549 RNase L −/− cells were infected with the indicated viruses (MOI = 3.0). One dpi, cell lysates were analyzed by immunoblotting with the indicated antibodies. Data are representative of two independent biological replicates.

Article Snippet: Membranes were blocked with 5 per cent non-fat milk dissolved in TBST (20 M Tris, 150 mM NaCl, 0.1 per cent Tween 20, pH 7.4) for 1 h and probed with one of the following primary antibodies: anti-PKR (sc-6282; Santa Cruz Biotechnology, Inc.), anti-phospho-PKR (ab32036; Abcam), anti-eIF2α or anti-phospho-eIF2α (Ser51) antibody (9722 and 9721, respectively; Cell Signaling Technology), anti-TRS1 999 , or anti-actin (A2066; Sigma).

Techniques: Infection, Agarose Gel Electrophoresis, Serial Dilution, Western Blot

Journal: Virus Evolution

Article Title: Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

doi: 10.1093/ve/veac105

Figure Lengend Snippet: p12 viruses inhibit both PKR and RNase L pathways in human cells. (A) A549 cells were infected with the indicated viruses (MOI = 3.0). One dpi, we collected either cell lysates (A) or total RNA (B). Cell lysates were analyzed by immunoblotting with the indicated antibodies (A). Total RNA was visualized on an agarose gel + 1 per cent bleach (B). White bars separate lanes that were moved to align with panel A; however, all lanes shown were run on the same gel.

Article Snippet: Membranes were blocked with 5 per cent non-fat milk dissolved in TBST (20 M Tris, 150 mM NaCl, 0.1 per cent Tween 20, pH 7.4) for 1 h and probed with one of the following primary antibodies: anti-PKR (sc-6282; Santa Cruz Biotechnology, Inc.), anti-phospho-PKR (ab32036; Abcam), anti-eIF2α or anti-phospho-eIF2α (Ser51) antibody (9722 and 9721, respectively; Cell Signaling Technology), anti-TRS1 999 , or anti-actin (A2066; Sigma).

Techniques: Infection, Western Blot, Agarose Gel Electrophoresis

Journal: Virus Evolution

Article Title: Gene amplification acts as a molecular foothold to facilitate cross-species adaptation and evasion of multiple antiviral pathways

doi: 10.1093/ve/veac105

Figure Lengend Snippet: Phenotypic changes to PKR and RNase L inhibition occur even after replication is fully rescued in human cells. (A) A549 cells were infected with the indicated viruses (MOI = 3.0). One dpi, we collected either cell lysates (top panel) or total RNA (bottom panel). Cell lysates were analyzed by immunoblotting with the indicated antibodies. Total RNA was visualized on an agarose gel + 1 per cent bleach. (B) A549 RNase L −/− cells were infected with the indicated viruses (MOI = 3.0). One dpi, cell lysates were analyzed by immunoblotting with the indicated antibodies. (C) A549 PKR −/− cells were infected with the indicated viruses (MOI = 3.0). One dpi, total RNA was harvested and visualized on an agarose gel + 1 per cent bleach. Data are representative of three independent biological replicates.

Article Snippet: Membranes were blocked with 5 per cent non-fat milk dissolved in TBST (20 M Tris, 150 mM NaCl, 0.1 per cent Tween 20, pH 7.4) for 1 h and probed with one of the following primary antibodies: anti-PKR (sc-6282; Santa Cruz Biotechnology, Inc.), anti-phospho-PKR (ab32036; Abcam), anti-eIF2α or anti-phospho-eIF2α (Ser51) antibody (9722 and 9721, respectively; Cell Signaling Technology), anti-TRS1 999 , or anti-actin (A2066; Sigma).

Techniques: Inhibition, Infection, Western Blot, Agarose Gel Electrophoresis

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Leptin transcriptional activation of CBF (or CSL) promoter is linked to several leptin-induced signaling pathways. (A) 4T1 cells were transiently transfected with a CBF-Luc reporter construct and incubated with leptin (0 and 1.2 nM) alone or plus siRNA oligonucleotides (SignalSilence control-siRNA and STAT3, MEK1, AKT1, mTOR, JNK, p38-siRNA). (B) Protein levels of kinases after siRNA treatment were determined by WB analysis using β-actin as loading control. (C) 4T1 cells transfected with CBF-Luc reporter were incubated with leptin alone or plus pharmacological inhibitors of JAK2/STAT3 (AG490, 30 µM), MEK/MAPK/ERK1/2 (PD98059, 30 µΜ), PI-3K/AKT1 (Wortmannin, 50 nM), PKC-Ca dependant (Gö6976, 30 µΜ), p38 kinase (SB203580, 2 µΜ), JNK (SP600125, 30 µM) and mTOR (Rapamycin, 20 nM) signaling pathways for 24 h. Luciferase activity was determined as described (see M &M) and expressed as a percent of basal or leptin-treated cells. (a) P<0.05 when comparing levels of luciferase activity to control (basal) or leptin-treated cells. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: STAT3, MEK1, AKT1, mTOR, SAPK/JNK and p38 MAPK-siRNA and, SignalSilence control siRNA were purchased from Cell Signaling (Danvers, MA).

Techniques: Activation Assay, Transfection, Construct, Incubation, Luciferase, Activity Assay, Derivative Assay

Journal: PLoS ONE

Article Title: Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

doi: 10.1371/journal.pone.0021467

Figure Lengend Snippet: Leptin transcriptional activation of CBF (or CSL) promoter is linked to several leptin-induced signaling pathways. (A) 4T1 cells were transiently transfected with a CBF-Luc reporter construct and incubated with leptin (0 and 1.2 nM) alone or plus siRNA oligonucleotides (SignalSilence control-siRNA and STAT3, MEK1, AKT1, mTOR, JNK, p38-siRNA). (B) Protein levels of kinases after siRNA treatment were determined by WB analysis using β-actin as loading control. (C) 4T1 cells transfected with CBF-Luc reporter were incubated with leptin alone or plus pharmacological inhibitors of JAK2/STAT3 (AG490, 30 µM), MEK/MAPK/ERK1/2 (PD98059, 30 µΜ), PI-3K/AKT1 (Wortmannin, 50 nM), PKC-Ca dependant (Gö6976, 30 µΜ), p38 kinase (SB203580, 2 µΜ), JNK (SP600125, 30 µM) and mTOR (Rapamycin, 20 nM) signaling pathways for 24 h. Luciferase activity was determined as described (see M &M) and expressed as a percent of basal or leptin-treated cells. (a) P<0.05 when comparing levels of luciferase activity to control (basal) or leptin-treated cells. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.

Article Snippet: STAT3, MEK1, AKT1, mTOR, SAPK/JNK and p38 MAPK-siRNA and, SignalSilence control siRNA were purchased from Cell Signaling (Danvers, MA).

Techniques: Activation Assay, Transfection, Construct, Incubation, Luciferase, Activity Assay, Derivative Assay