Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 1 mTORC2 affects rearrangement of the F-actin cytoskeleton in mouse fertilized eggs. (a) The development model of mouse fertilized eggs. G1 phase: 12–21 hours after injecting hCG; S phase: 21–27 hours after injecting hCG; G2 phase: 27–30 hours after injecting hCG; M phase: 30–33 hours after injecting hCG. (b) Staining for F-actin (red) and DNA (blue) revealed the organization of the F-actin cytoskeleton in mouse fertilized eggs transfected with RICTOR shRNA (F-actin: yellow arrow). Scale bar: 20 μm. (c) Western immunoblotting detection of mTORC2 in 200 mouse fertilized eggs treated with shRNA targeted against mTORC2. WB: Western blotting

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Staining, Transfection, shRNA, Western Blot

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 2 Akt1 regulates cell division and F-actin rearrangement in mouse fertilized eggs. (a) Western immunoblotting detection of Akt1 in 200 mouse fertilized eggs treated with siRNA targeted against Akt1. WB: Western blotting. (b) The division rate in cultured mouse embryos after 0.03 ng of mRNA-encoding Akt1-WT, myr-Akt1, Akt1-KD or Akt1 siRNA injection. The percentage of cells undergoing cell division and cell survival was calculated after manual counting under a dissecting microscope 35 hours after injection of human chorionic gonadotropin in female mice. The numbers of eggs undergoing cell division (hatched bars) or survival are given above each bar graph. (c) Cortical remodelling of F-actin is induced by Akt1 activation. Mouse fertilized eggs were stained with rhodamine-phalloidin (red fluorescence) to visualize F-actin cytoskeleton. Various scenarios were studied, which included cells injected with mRNA-encoding Akt1-WT, myr-Akt1, Akt1-KD or siRNA targeted against Akt1. Fertilized eggs were fixed and labelled with rhodamine-phalloidin (10 or 20 μmol/L; F-actin labelling, indicated by white arrows) and with Hoechst 33258 (1 mg/ml; DNA labelling) and imaged by laser-scanning confocal microscope. Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Western Blot, Cell Culture, Injection, Microscopy, Activation Assay, Staining, Fluorescence

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 4 Girdin is essential for rearrangement of the F-actin cytoskeleton and the development of mouse fertilized eggs. (a) Depletion of Girdin in mouse one-cell staged fertilized eggs by siRNA. Total cell extracts from control siRNA- and Girdin siRNA- injected one-cell staged fertilized eggs were subjected to Western blot analyses and immunodetection with anti-Girdin, anti-p-Girdin, anti-Akt1 and anti-actin antibodies. (b) The cleavage rate in cultured mouse embryos after Girdin siRNA injections shows that the total number of eggs undergoing cell division is given above each bar graph from three independent experiments. (c) F-actin cytoskeleton of embryos derived from fertilized eggs treated with Girdin siRNA. Mouse fertilized eggs were treated with control or Girdin siRNAs and fixed 48 hours later, followed by staining with rhodamine-phalloidin and anti-Girdin antibody. In situ validation of the interaction between Girdin and polymerized F-actin by confocal microscopy is shown. One-cell stage mouse fertilized eggs were treated with 10 or 20 μmol/L Girdin siRNA. Immunolocalization of Girdin is revealed by green staining (antibody), and immunolocalization of actin is revealed by red staining. Control fertilized eggs (21 hours after hCG): one-cell arrested embryos derived from fertilized eggs that were injected with Girdin siRNA 21 hours after hCG and cultured for 24 hours; control two-cell embryo: in control fertilized eggs, F-actin forms a regular ring in the cell cortex. In embryos derived from fertilized eggs treated with girdin siRNA, polymerized F-actin was observed in the cytoplasm and formed irregular patches that were scattered randomly in the cortex. Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Control, Injection, Western Blot, Immunodetection, Cell Culture, Derivative Assay, Staining, In Situ, Biomarker Discovery, Confocal Microscopy

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 3 The mTORC2/Akt1 pathway rearranges the F-actin cytoskeleton of one-cell stage fertilized eggs. Microinjection of Rictor shRNA then with myr-Akt1 mRNA into mouse one-cell stage embryos. Staining for F-actin (red) revealed the organization of the F-actin cytoskeleton in mouse fertilized eggs (F-actin is shown by the yellow arrow). Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Microinjection, shRNA, Staining

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 5 The Akt1/ Girdin pathway regulates rearrangement of the F-actin cytoskeleton of one-cell stage fertilized eggs. (a) One-cell stage mouse embryos were first microinjected with Akt1-WT mRNA, myr-Akt1 mRNA or siRNA, then stained with rhodamine-phalloidin (20 μmol/L; actin labelling as shown in red) and anti-P-Girdin (1:50; Girdin labelling; as shown in green). Merged colour detection between P-Girdin and polymerize F-actin appears as yellow stained images. The areas of co-localization are highlighted with yellow arrowheads. Scale bar: 20 μm. (b) 200 fertilized eggs were treated with mRNA coding for Akt1- WT, myr-Akt1, Akt1-KD or siRNA against Akt1. Western immunoblot analyses assessed following immunoreaction with anti-P-Girdin (upper panel) and anti-Girdin (lower panel) antibodies are shown. (c) One-cell stage mouse embryos were first microinjected with 0.03 ng of myr-Akt1 mRNA as described in the Materials and Methods, and then 1–2 hours later with Girdin siRNA, following which specimens were stained with rhodamine-phalloidin (20 μmol/L; actin labelling shown in red). Scale bar: 20 μm

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Staining, Western Blot

Journal: Cell proliferation

Article Title: F-actin rearrangement is regulated by mTORC2/Akt/Girdin in mouse fertilized eggs.

doi: 10.1111/cpr.12285

Figure Lengend Snippet: FIGURE 6 The mTORC2/Akt1/Girdin pathway rearranges the F-actin cytoskeleton in one-cell stage fertilized eggs. (a) The effect of gene knockdown on the expression of Rictor and the phosphorylation of Girdin-1417. Extracts of mouse fertilized eggs were resolved by 6% SDS-PAGE, transferred to nitrocellulose and probed with phosphor- Girdin-Ser1417 antibody and Girdin antibody. (b) We examined Girdin phosphorylation in the fertilized eggs treated with Rictor shRNA, WT-Akt1 mRNA, Rictor shRNA and WT-Akt1 coinjection. In WT-Akt1 mRNA treated cells, we noted that Girdin was highly phosphorylated at 1417. But cells treated with Rictor siRNA and WT-Akt1 exhibited decreased phosphorylation of Girdin 1417

Article Snippet: The constructs of Rictor shRNA were provided by Dr. Estella Jacinto (University of Basel, Switzerland). siRNA for mouse Girdin was purchased from Santa Cruz (Girdin siRNA sc- 145407 CA, USA). siRNA for mouse Akt1 was purchased from Santa Cruz (Akt1 siRNA (m): sc- 29196 CA, USA).

Techniques: Knockdown, Expressing, Phospho-proteomics, SDS Page, shRNA

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: TRAF3IP2 mediates high glucose-induced endothelin-1 production as well as endothelin-1-induced inflammation in endothelial cells

doi: 10.1152/ajpheart.00478.2017

Figure Lengend Snippet: High glucose (HG) induces activation of activator protein 1 (AP-1) in part via TRAF3 interacting protein 2 (TRAF3IP2) and JNK in human aortic endothelial cells (HAECs). A: HG (25 mM d-glucose) induces time-dependent TRAF3IP2 expression. At 70% confluency, the complete medium on HAECs was replaced with endothelial basal medium-2 (without supplements) for 2 h, and cells were then incubated with HG for the indicated time periods and analyzed for TRAF3IP2 protein expression by immunoblot analysis. B: HG (15 and 25 mM), but not mannitol, induces TRAF3IP2 expression. HAECs treated as in A with HG or mannitol for 30 min were analyzed for TRAF3IP2 by immunoblot analysis. C: HG (25 mM) induces time-dependent JNK activation. HAECs treated as in A with HG were analyzed for JNK activation using activation-specific antibodies. Total JNK served as a control. D: HG (25 mM) induces JNK activation via TRAF3IP2. At 50% confluency, HAECs were infected with lentiviral TRAF3IP2 or control enhanced green fluorescent protein (eGFP) shRNA (multiplicity of infection 0.5 for 48 h). Cells were then treated with HG for 60 min. Total and activated JNK were analyzed as in C. Knockdown of TRAF3IP2 was confirmed by immunoblot analysis and is shown on the right. ASK1 served as an off-target control. E: HG (25 mM) induced AP-1 activation via TRAF3IP2 and JNK. HAECs infected with TRAF3IP2 or JNK1 shRNA or pretreated with the JNK inhibitor SP600125 (20 µM for 30 min) were incubated with HG for 60 min and analyzed for AP-1 activation by immunoblot analysis using antibodies that specifically detect phosphorylated c-Jun at Ser73. Knockdown of JNK1 is shown in the top right. Bar graphs in A–E represent densitometric analyses from 3 independent experiments. *P ≤ 0.05 vs. control (i.e., open bar); †P ≤ 0.05 vs. HG (n = 3).

Article Snippet: The source and concentration of antibodies used in the immunoblot analysis were as follows: TRAF3IP2 (1 μg/ml, IMG-563, Novus Biologicals, Littleton, CO), tubulin (1:1,000, no. 2144, Cell Signaling Technology, Beverly, MA), phospho-JNK (Thr 183 /Tyr 185 , 1:1,000, no. 9251, Cell Signaling Technology), JNK (1:1,000, no. 9252, Cell Signaling Technology), phospho-c-Jun (Ser 63 , 1:1,000, no. 9261, Cell Signaling Technology), c-Jun (1:1,000, no. 9165, Cell Signaling Technology), apoptosis signal-regulating kinase 1 (ASK1) (1:1,000, no. 8622, Cell Signaling Technology), Akt (1 μg/ml, no. 9272, Cell Signaling Technology), phospho-IKKβ (1:1,000, no. 2697, Cell Signaling Technology), IKKβ (1:1,000, no. 2370, Cell Signaling Technology), phospho-p65 (1:1,000, no. 3033, Cell Signaling Technology), p65 (1:1,000, no. 3034, Cell Signaling Technology), ECE1 (1 μg/ml, ab71829, Abcam, Cambridge, MA), ICAM-1 (1:400, sc-8439, Santa Cruz Biotechnology), VCAM-1 (1:200, sc-8304, Santa Cruz Biotechnology), and cleaved caspase-3 (1 μg/ml, no. 9664, Cell Signaling Technology).

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Control, Infection, shRNA, Knockdown

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Rictor Deficiency Aggravates Hepatic Ischemia/Reperfusion Injury in Mice by Suppressing Autophagy and Regulating MAPK Signaling.

doi: 10.1159/000488165

Figure Lengend Snippet: Fig. 7. Rictor deficiency affects MAPKs activation on LPS treat ment RAW264.7 cell line. (A) Western blot analysis of Rictor, P-JNK, JNK, P-ERK and ERK in control and Rictor siRNA pretreat ment of RAW264.7 at the indicated time points, upon LPS stimuli. (B) Supernatant cytokine levels of IL-6 and TNF-a after in LPS-stimulated at the indicated time points, mea sured by ELISA. (C) mRNA levels of cytokines and chemokines such as IL-6, TNF-a, IL-1β and IL-10 were determined by quantitative real- time PCR. n=3-5 replicates per ex periment group performed on dif ferent days. All data shown as the mean ± SEM, **P<0.01, *P<0.05.

Article Snippet: RICTOR-targeting shRNA plasmids (sc-61479-SH) and control shRNA plasmids (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: microRNA-153 Targets mTORC2 Component Rictor to Inhibit Glioma Cells

doi: 10.1371/journal.pone.0156915

Figure Lengend Snippet: Relative Rictor mRNA (A) and miRNA-153 (C) expression in stable U87MG cells with scramble control shRNA (“shRNA-C”) or Rictor shRNA (“shRNA-Rictor”) was tested by Real-time PCR assay. Expressions of listed proteins in these cells were also shown (B). Same number of U87MG cells with “shRNA-C” or “shRNA-Rictor” were subjected to MTT assay (D) and clonogenicity assay (E) to test cell growth; Cell apoptosis was also tested (Histone DNA ELISA assay, F). Rictor shRNA-expressing stable U87MG cells were transfected with miR-153 or microRNA-control (“miR-C”), miR-153 expression (G, Real-time PCR assay), cell growth (E, MTT assay) and apoptosis (F, Histone DNA ELISA assay) in these cells were tested. Rictor expression (vs. Tubulin) and p-Akt Ser473 (vs. Akt1) were quantified (B). Bars stand for mean ± SD. * p < 0.05 vs. “shRNA-C” group (A, C, D-F, n = 5). * p < 0.05 vs. “miR-C” group (G, n = 5).

Article Snippet: The Rictor shRNA lentiviral particles were purchased from Santa Cruz Biotech (sc-61478-V, Santa Cruz, CA).

Techniques: Expressing, shRNA, Real-time Polymerase Chain Reaction, MTT Assay, Enzyme-linked Immunosorbent Assay, Transfection