Journal: Oncotarget

Article Title: TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

doi: 10.18632/oncotarget.20429

Figure Lengend Snippet: (A) Representative images of IHC staining of low (left panel) and high (right panel) TSSC3 expressing cells. (B) Kaplan-Meier curve showing that higher expression of TSSC3 is significantly associated with a better prognosis (P < 0.05). (C) TSSC3 expression in monolayer MTH or SaOS2 cells is higher than that of MTH or SaOS2 sphere cells, respectively (Bars, mean±SEM, *P < 0.05. Upper panel, RT-PCR; lower panel, Western blot). (D) The number of CD133, CD117, and Stro-1 positive MTH (left) or SaOS2 cells (right) significantly decreased after overexpression of TSSC3 (Bars, mean±SEM, * P < 0.05). (E) Efficiency of tumor sphere formation by Lv-TSSC3 MTH or Lv-TSSC3 SaOS2 cells is lower than that of Lv-empty MTH and SaOS2 cells, respectively (Bars, mean±SEM, *P < 0.05). (F) Nanog expression is reduced after TSSC3 overexpression in MTH (left) or SaOS2 (right) cells; meanwhile, Oct4 and Sox2 expression levels were slightly decreased. (G) Nanog expression is suppressed after overexpression of TSSC3 (Bars, mean±SEM, *P < 0.05). (H) There are significantly fewer xenografts generated by Lv-TSSC3 MTH than by Lv-empty MTH cells (N=4; P = 0.0028).

Article Snippet: Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China).

Techniques: Immunohistochemistry, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Generated

Journal: Oncotarget

Article Title: TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

doi: 10.18632/oncotarget.20429

Figure Lengend Snippet: (A) Representative IHC staining images of low (left panel) and high (right panel) Nanog expression. (B) Kaplan-Meier curve showing that higher expression of Nanog is significantly related to a poor prognosis ( P < 0.05). (C) Efficiency of tumor sphere formation by Lv-TSSC3/Lv-Nanog MTH (left) or SaOS2 cells (right) is higher than in Lv-TSSC3/Lv-empty cells, respectively (Bars, mean±SEM, * P < 0.05). (D) The percentage of CD133, CD117 and Stro-1 positive Lv-TSSC3 MTH (left) or SaOS2 cells (right) is significantly increased after Nanog overexpression (Bars, mean±SEM, * P < 0.05). (E) The IC 50 values of Lv-TSSC3 MTH and SaOS2 cells under cisplatin treatmentis are higher after Nanog overexpression. Migration (F) and invasion (G) capacity is enhanced in Lv-TSSC3 MTH and SaOS2 cells after Nanog overexpression (Bars, mean±SEM, * P < 0.05). (H) There are significantly more xenografts generated by MTH and SaOS2 cells after Nanog overexpression (N=5; P = 0.0001; P = 0.004).

Article Snippet: Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China).

Techniques: Immunohistochemistry, Expressing, Over Expression, Migration, Generated

Journal: Oncotarget

Article Title: TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

doi: 10.18632/oncotarget.20429

Figure Lengend Snippet: (A) Overexpression of TSSC3 inhibits Src/Akt pathway activity in OS cells. RT-PCR (B) and Western blot analysis (C) show that inactivation of Src results in reduced Nanog expression in OS cells (Bars, mean±SEM, * P < 0.05). (D) Knockdown of Src results in reduced Nanog expression in MTH (left) and SaOS2 cells (right; Bars, mean±SEM, * P < 0.05), while overexpression of TSSC3 does not further reduce Nanog expression (Bars, mean±SEM, P > 0.05). (E) The number of CD133, CD117 and Stro-1 positive MTH (left) and SaOS2 cells (right) is significantly decreased after Src knockdown (Bars, mean±SEM, * P < 0.05), while overexpression of TSSC3 only slightly further decreases expression of these markers. (F) Efficiency of tumor sphere formation by Lv-empty/siSrc MTH (left) and SaOS2 cells (right) is lower than that of scrambled MTH and SaOS2 cells (Bars, mean±SEM, * P < 0.05), while overexpression of TSSC3 does not have a significant additional influence on sphere formation. (G) Tumor spheres formed by Lv-empty/siSrc MTH (upper three images, the scale bar is 100 μm) or SaOS2 cells (lower three images, the scale bar is 100 μm) are smaller in size than those formed by scrambled/Lv-empty MTH or SaOS2. Overexpression of TSSC3 does not have a significant influence on the size of tumor spheres in siSrc cell lines.

Article Snippet: Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China).

Techniques: Over Expression, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Knockdown

Journal: Oncotarget

Article Title: TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

doi: 10.18632/oncotarget.20429

Figure Lengend Snippet: RT-PCR (A) and Western blot analysis (B) show that inactivation of Akt results in reduction of Nanog in OS cells (Bars, mean±SEM, * P < 0.05). (C) Overexpression of TSSC3 inactivates the Akt pathway, and decreases Nanog expression in MTH (left) and SaOS2 cells (right). Treatment with IGF1 activates the Akt pathway and increases Nanog expression. (D) Efficiency of tumor sphere formation by Lv-TSSC3 MTH (left) or SaOS2 cells (right) is lower than that of Lv-empty MTH and SaOS2 cells. IGF1 treatment enhances the efficiency (Bars, mean±SEM, * P < 0.05). (E) The number of CD133, CD117 and Stro-1 positive Lv-TSSC3 MTH (left) or SaOS2 cells (right) significantly decreases compared to Lv-empty cells. IGF1 treatment significantly enhances the number of marker positive cells (Bars, mean±SEM, * P < 0.05).

Article Snippet: Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Over Expression, Expressing, Marker

Journal: Oncotarget

Article Title: TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

doi: 10.18632/oncotarget.20429

Figure Lengend Snippet: (A) Representative images of IHC staining for low (left) and high (right) p-Src expression levels. (B) Kaplan-Meier curve showing that lower expression of p-Src is significantly associated with an improved prognosis ( P < 0.05). (C) Scores of IHC staining for p-Src and p-Akt are positively associated (upper panel, Bars, mean±SEM, * P < 0.05); meanwhile, those for p-Src and Nanog are also positively associated (lower panel, Bars, mean±SEM, * P < 0.05). (D) Schematic summarizing our findings. In OS cells, higher Nanog expression significantly enhances stemness, motility and chemoresistance. TSSC3 inhibits Src activity, resulting in decreased activity of the Akt pathway and leading to a reduction in Nanog expression and stem-like phenotype in OS cells.

Article Snippet: Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China).

Techniques: Immunohistochemistry, Expressing, Activity Assay

Journal: Oncotarget

Article Title: TSSC3 represses self-renewal of osteosarcoma stem cells and Nanog expression by inhibiting the Src/Akt pathway

doi: 10.18632/oncotarget.20429

Figure Lengend Snippet: Correlation between IHC staining and pathological parameters in osteosarcoma patients

Article Snippet: Primary antibodies included rabbit anti-human TSSC3 (1:1000; Novus biologicals), rabbit anti-human Nanog, Oct4, Sox2 (1:500; Cell Signaling Technology, Danvers, MA), rabbit anti-human Akt, p-Akt, Src, p-Src (1:1000; Cell Signaling Technology), mouse anti-human CD44 antibody (1:500; Cell Signaling Technology), mouse anti-human CXCR4 antibody (Abcam, Cambrige, MA, USA) and rabbit anti-human GAPDH (1:1000; Biodragon Immunotech, China).

Techniques: Immunohistochemistry

Journal: Journal of Lipid Research

Article Title: Naturally occurring variant of mouse apolipoprotein A-I alters the lipid and HDL association properties of the protein

doi: 10.1194/jlr.M021154

Figure Lengend Snippet: T7-ApoA-I binding to J774 macrophages with and without CPT-cAMP induction of ABCA1 expression. J774 macrophages were incubated 15 h with CPT-cAMP to induce ABCA1 expression. C57 or FVB recombinant T7-ApoA-I was then incubated with cells for 2 h and removed by PBS wash. A: T7-ApoA-I binding was measured by western blot of the T7-ApoA-I remaining associated with the cell protein as described in Materials and Methods. B: ABCA1 levels postincubation with 2 μg/ml C57 or FVB recombinant T7-ApoA-I as measured by ABCA1 Western blot of cell protein as described in Materials and Methods. Data points show the mean ± SD of triplicate samples.

Article Snippet: ABCA1 levels from the same samples were run on a 4–12% gradient SDS-PAGE, transferred to Immobilon, and probed with rabbit-anti-mouse ABCA1 antibody (Novus Biologicals, Littleton, CO). rHDL cholesterol esterification assay rHDL was generated from C57 or FVB T7-ApoA-I using phosphatidylcholine, T7-ApoA-I, 3 H-cholesterol, and nonlabeled cholesterol (molar ratio 200:1.2:1:1).

Techniques: Binding Assay, Expressing, Incubation, Recombinant, Western Blot

Journal: The Journal of Neuroscience

Article Title: Regulation of Nociceptive Glutamatergic Signaling by Presynaptic Kv3.4 Channels in the Rat Spinal Dorsal Horn

doi: 10.1523/JNEUROSCI.3212-17.2018

Figure Lengend Snippet: Intact cervical spinal cord preparation for patch-clamp recordings of glutamatergic synaptic currents from the superficial dorsal horn. A, Schematic of the experimental setup representing its main components. Neurons in the superficial dorsal horn are visualized using oblique infrared LED illumination and a 40× immersion objective. The spinal cord (SC) was pinned at an angle of 10° to 15° on a piece of elastomer compound eraser. The spinal cord is represented by a cross-section of the cervical region with its axis perpendicular to the plane of the image. BSE, Bipolar stimulation electrode (suction electrode); DR, dorsal roots (one free and the other inside the suction electrode); PCE, patch-clamping electrode hooked up to a Multiclamp 700B amplifier. B, Images of lamina I neurons subjected to whole-cell patch clamping. Top, Infrared image. Bottom, Fluorescence image of neuron loaded with biocytin (conjugated with Alexa Fluor 488) through the PCE. C, Representative monosynaptic eEPSCs evoked consecutively by stimulating the DR (100 μA, 1 ms, 10 sweeps) while holding the neuron's VH at −70 mV (see Materials and Methods). The average trace is shown in black. D, Histogram of eEPSC peak amplitudes. The stimulus intensity ranged between 100 and 600 μA. E, Consecutive eEPSCs recorded before and after exposing the spinal cord to 1 μm CNQX (averages are displayed in black and red, respectively). F, Spontaneous glutamatergic synaptic currents at VH = −70 mV before (black) and after (red) exposure to 1 μm CNQX.

Article Snippet: Sections were then washed 3 times (5 min each) and incubated with goat anti-rabbit Alexa Fluor 488 (1:150; Abcam) or goat anti-guinea pig Alexa Fluor 568 (1:500; Thermo Fisher Scientific) in 5% NGS in PBST for 1 h at room temperature.

Techniques: Patch Clamp, Fluorescence

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: (A) Characterization of anti-phospho-eEF-2K (Ser-500) antibody. The antibody was characterized against 50 ng recombinant eEF-2K by immunoblotting as described under ‘Experimental Procedures’. Lanes: 1 – untreated eEF-2K WT; 2 – autophosphorylated eEF-2K WT; 3 – untreated eEF-2K S500A; 4 – autophosphorylated eEF-2K S500A; 5 – untreated eEF-2K S500D; 6 – autophosphorylated eEF-2K S500D. (B) Time course of incorporation of phosphate at Ser-500. eEF-2K (500 nM) was allowed to autophosphorylate in the presence of 5 µM CaM and 50 µM free Ca2+. At the indicated times, 50 ng of eEF-2K were removed and the reaction quenched with hot SDS-PAGE sample loading buffer. The samples were then analyzed by Western blotting using the anti-phospho-eEF-2K (Ser-500) antibody as described under ‘Experimental Procedures’. (C) Graphical representation of (B). Western blots were quantified using ImageJ, and data then plotted as the percent phosphorylation of Ser-500 against autophosphorylation time. Inset: Expansion of the data for 0–70 min. Experiments were performed in duplicate, and error bars represent the standard deviation. (D) Buffers used are described under ‘Experimental Procedures’. Assays were performed with eEF-2K enzyme, ± 50 µM free Ca2+ and ± 2 µM calmodulin. EGTA (1 mM) was added to all assays conducted in the absence of Ca2+. For eEF-2K WT, S500A and S500D assayed in the presence of both Ca2+ and CaM, and eEF-2K S500D assayed in the presence of only CaM, activities were much higher than the basal level of kinase activity, and hence only 5 nM of kinase was used. For all the other assays, 50 nM eEF-2K was used in order to detect an increase in kinase activity over the basal level. Kinase activity was determined by measuring the rate of phosphorylation of the peptide (µM.s−1). Activities of the mutants are reported as the percentage of the wild type activity.

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Recombinant, Western Blot, SDS Page, Standard Deviation, Activity Assay

Journal: Biochemistry

Article Title: Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 to Regulate its Activity and Calcium Dependence

doi: 10.1021/bi201788e

Figure Lengend Snippet: Summary of the various phosphorylated residues on eEF-2K. Components are color coded as follows: ( - red) – suggested to be involved in the negative regulation of eEF-2K activity through an inhibitory phosphorylation (these sites include Ser-78, Ser-359, Ser-366 and Ser-396). Regulation through the mTOR pathway involves the phosphorylation of Ser-366 by p70 S6 kinase, and the phosphorylation of Ser-359 and Ser-78 by at least two additional unknown kinases (22–24). It has been postulated that the Ser-78 phosphorylation acts to hinder the binding of CaM to eEF-2K (24). The cdc2-cyclin B complex has been shown to modulate eEF-2K activity via Ser-359 in a manner that is dependent on the cell cycle as well as amino acid availability, and is perhaps controlled by mTOR (25). Regulation through the MAPK cascade occurs via the phosphorylation of Ser-366 by p90RSK1 in an ERK-dependent fashion (22). In addition, the stress-activated protein kinases p38α and p38δ inhibit eEF2K via phosphorylation on Ser-396 (23). p38δ is also known to phosphorylate eEF-2K on Ser-359 (21); ( - green) – suggested to be involved in the positive regulation of eEF-2K activity through an activating phosphorylation (these sites include Ser-398 and Ser-500). Phosphorylation of Ser-398 by the energy-supply regulator AMPK is known to activate eEF-2K (29). The cAMP-dependent PKA has also been shown to activate eEF-2K via a phosphorylation on Ser-500, and in the process imparts Ca2+-independent activity to the kinase (26–28); ( - blue) – involved in autophosphorylation of eEF-2K (these sites include Thr-348, Thr-353, Ser-445, Ser-474 and Ser-500). Of the 5 autophosphorylation sites, only Thr-348 appears to be essential for activity against its substrate. Ser-500 is an autophosphorylation site and is also known to be phosphorylated by PKA, and could be the key residue responsible for autophosphorylation-induced Ca2+-independent (CaM-dependent – this work) activity (16, 17). The role of the phosphorylation at Ser-377 by MAPKAP-K2 has not yet been determined (23).

Article Snippet: Monitoring incorporation of phosphate at Ser-500 by immunoblotting To analyze the time course of phosphate incorporation at Ser-500, ECM Biosciences generated affinity-purified rabbit polyclonal anti-eEF-2K (Ser-500) phospho-specific antibodies, which were used in Western blotting.

Techniques: Activity Assay, Binding Assay