Journal: Frontiers in Immunology

Article Title: ACSL1 Inhibits ALV-J Replication by IFN-Ⅰ Signaling and PI3K/Akt Pathway

doi: 10.3389/fimmu.2021.774323

Figure Lengend Snippet: ACSL1 induced apoptosis via PI3K/Akt signaling pathway. (A, B) ACSL1 overexpression (A) and knockdown (B) in MDMs for 48 h, followed by infection with ALV-J (10 4 TCID 50 /0.1 ml) before assays. Apoptosis analysis of MDMs for 3 and 6 hpi, using Annexin V-FITC. Statistical analysis of the data from the multiple repeated Annexin V-FITC experiments. (C, D) Immunoblot analysis of the levels of env, Akt, p-Akt, mTOR, p-mTOR, IKKα/β, and p-IKK expression in MDMs transfected with ACSL1 expression plasmids or empty vector control for 48 h followed by ALV-J infection for 3 h. (E, F) Immunoblot analysis of the levels of env, Akt, p-Akt, mTOR, p-mTOR, IKKα/β, and p-IKK expression in MDMs transfected with siACSL1 or control siRNA for 48 h followed by ALV-J infection for 3 h . (G, H) qRT-PCR analysis of apoptosis-related genes ( AIF , CYCS , FKHR , MDM2 , and caspase-9 ) in ACSL1 overexpressed cells (G) or ACSL1 knockdown cells (H) followed by ALV-J infection for 3 h. Data shown are the means ± SEM (n=3). P values were calculated using two-tailed unpaired Student’s t-test. Differences with P < 0.05 were considered significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-ALV-J envelope protein JE9 (kindly provided by Dr. Aijian Qin, Yangzhou University, Yangzhou, China), anti-Flag (AF5051, Beyotime), FITC-labeled goat anti-rabbit IgG (H+L) (A0562, Beyotime), anti-STAT1 (70R-51641, Fitzgerald), anti-phosphorylated STAT1 (15H13L67, Invitrogen), anti-Akt (10176-2-AP, Proteintech), anti-phosphorylated Akt (D9E, Cell Signaling), anti-mTOR (bs-1992R, Bioss), anti-phosphorylated mTOR (D9C2, Cell Signaling), anti-IKKα/β (bs-10123R, Bioss), anti-phosphorylated IKK (bs-3229R, Bioss), anti-β-actin (AF5003, Beyotime), Goat anti-rabbit IgG/HRP (bs-0295G, Bioworld), Goat anti-mouse IgG/HRP (bs-0296G, Bioworld).

Techniques: Over Expression, Infection, Western Blot, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Two Tailed Test

Journal: PLoS ONE

Article Title: Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling

doi: 10.1371/journal.pone.0078632

Figure Lengend Snippet: AsPC-1 (A) and MiaPaCa2 (B) cells were treated for 72 hr with 25 nM LP-1, LP-4, non-targeting siRNA (nt), or a mock transfectant (m). Western blots were done with the indicated antibodies to mTOR effector proteins and mTOR-specific phosphorylation sites, as well as a Vinculin loading control. The graphs show densitometric quantitation of the phosphorylated bands normalized to both Vinculin and their corresponding whole protein. Results are representative of three independent experiments.

Article Snippet: Equal amounts of lysates were immunoprecipitated against mTOR antibody (1:350 v/v) overnight at 4°C then proteins were pulled down with Protein A agarose beads (Santa Cruz Biotechnology) for an additional 2 hours at 4°C.

Techniques: Transfection, Western Blot, Phospho-proteomics, Control, Quantitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: PPT1 Reduction Contributes to Erianin-Induced Growth Inhibition in Oral Squamous Carcinoma Cells

doi: 10.3389/fcell.2021.764263

Figure Lengend Snippet: Erianin decreases PPT1 expression and affects mTOR signaling in OSCC cells. Tumor cells were plated into 6-well or 12-well plates and treated as described. (A–B) The expression of PPT1 and mTOR signaling-related proteins in OSCC cells with or without erianin treatment were determined. (C) The PLA assay was performed to determine the interaction between mTOR and RHEB. (Scale bars, 10 μm).

Article Snippet: Antibodies for Cyto C, caspase 9, cleaved caspase 3, GSDMD, CDC25C, CDC2, phospho-CDC2 ( p -CDC2, Thr161), cyclin B1 and phospho-4EBP1 (p-4EBP1) were obtained from Cell Signaling Technology (Danvers, MA, United States); antibodies for PARP, NLRP3, 4EBP1, mTOR, phospho-mTOR ( p -mTOR), RHEB, LC3B, beclin-1 and p62 were obtained from Proteintech Group (Wuhan, China); antibodies for cleaved-caspase 1, GSDME and PPT1 were from Abcam (Cambridge, MA, United States); and the antibody for β -actin was from Sigma-Aldrich.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: PPT1 Reduction Contributes to Erianin-Induced Growth Inhibition in Oral Squamous Carcinoma Cells

doi: 10.3389/fcell.2021.764263

Figure Lengend Snippet: In vivo anti-OSCC effects of erianin. The xenograft model was established, and mice were grouped and treated as described previously. (A) Graphs representing the average tumor volumes of the xenografts. (B) Representative images of the xenografts. (C) Weights of the tumors obtained from mice treated with or without erianin. (D) Body weights of the mice were recorded. (E) Serum aminotransferases levels were measured. (F) PPT1 and p-mTOR expression in the xenografts. (G) Integrated optical density values of the IHC staining. (Scale bars, 100 μm) Five mice were included for each group, and one representative experiment out of three is shown. (* p < 0.05, *** p < 0.001).

Article Snippet: Antibodies for Cyto C, caspase 9, cleaved caspase 3, GSDMD, CDC25C, CDC2, phospho-CDC2 ( p -CDC2, Thr161), cyclin B1 and phospho-4EBP1 (p-4EBP1) were obtained from Cell Signaling Technology (Danvers, MA, United States); antibodies for PARP, NLRP3, 4EBP1, mTOR, phospho-mTOR ( p -mTOR), RHEB, LC3B, beclin-1 and p62 were obtained from Proteintech Group (Wuhan, China); antibodies for cleaved-caspase 1, GSDME and PPT1 were from Abcam (Cambridge, MA, United States); and the antibody for β -actin was from Sigma-Aldrich.

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: Breast Cancer : Targets and Therapy

Article Title: The role of the chemokine receptor XCR1 in breast cancer cells

doi: 10.2147/BCTT.S126184

Figure Lengend Snippet: Activation of MAPK and PI3K/AKT/mTOR pathways is involved in XCR1-mediated tumor cell. Notes: ( A , C ) Western blot analysis of MAPK and PI3K/AKT/mTOR pathways protein expression in 231/vector and 231/XCR1 cells. ( B , D ) Phospo-P53 and LC3 protein expression were detected by immunohistochemistry analysis in xenografts of 231/vector and 231/XCR1 tumor bearing mice. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase.

Article Snippet: Antibodies against MEK1/2 (9126, 1:1000), Phospho-MEK1/2 (2338, 1:1000), Phospho-ERK1/2 (4376, 1:1000), AKT (4691.1:1000), Phospho-AKT (4060P,1:1000), Phospho-P38 (4511.1:1000), Phospho-JNK (4668,1:1000), E-cadherin (3195,1:1000), Phospho-p53 (9284.1:1000), and mTOR Substrates Sampler kit (CST 9862) that conclude Phospho-mTOR, mTOR, Phospho-p70s6389 (Thr389), Phospho-P70389 (Thr371), Phospho-4EBP1 (Thr37/46), and anti-rabbit IgG were from Cell Signaling Technology.

Techniques: Activation Assay, Western Blot, Expressing, Plasmid Preparation, Immunohistochemistry

Journal: American Journal of Cancer Research

Article Title: NRSN2 promotes osteosarcoma cell proliferation and growth through PI3K/Akt/MTOR and Wnt/β-catenin signaling

doi:

Figure Lengend Snippet: NRSN2 regulates PI3K/Akt/GSK3β axis and Wnt/β-catenin signaling in osteosarcoma cells. A. The level of phosphorylated Akt, mTOR, p-GSK3β and nuclear β-catenin (nu-β-catenin) are positively correlated with the level of NRSN2 in U2OS and MG63 cells. B. Luciferase reporter assays revealed that NRSN2 could regulate Wnt/β-catenin signaling in U2OS and MG63 cells. C. Knockdown NRSN2 inhibits the expression of CCND1 and c-myc in U2OS cells. D. Overexpression of NRSN2 elevates the mRNA levels of CCND1 and c-myc in MG63 cells. E. The pro-proliferation effect was reversed when treated with IWR-1-endo, a inhibitor of β-catenin. *p<0.05, **p<0.01.

Article Snippet: The following antibodies were used in this study: NRSN2 (1:1000, Proteintech), GAPDH (1:5000, Proteintech), p-Akt, Akt, p-mTOR, mTOR, p-GSK3β, GSK3β, β-catenin and the second antibodies were purchased from Cell Signaling Technology and with 1:1000 dilutions.

Techniques: Luciferase, Expressing, Over Expression