Journal: bioRxiv

Article Title: RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

doi: 10.1101/856880

Figure Lengend Snippet: NSB Protein Levels following Cellular Stress. Total protein extracts were prepared from untreated cells, heat shocked cells (42°C for 2 hours), and cells treated with the genotoxic stressor mitoxantrone (MTX; 20 µM for 6 hours). Each panel shows the results of loading 10 μg of each extract and blotting for the indicated proteins with actin used as a loading control. (A) RBM45; (B) HSF1; (C) SAFB. No statistically significant differences in the levels of the indicated proteins were detected between conditions ( p > 0.05).

Article Snippet: The following primary antibodies and dilutions were used for immunofluorescence and immunohistochemistry: rabbit polyclonal anti-RBM45 (Sigma, 1:75), rabbit polyclonal anti-RBM45 (custom; Pacific Immunology, 1:100), mouse monoclonal anti-scaffold attachment factor B (SAFB, Lifespan Biosciences, Seattle, WA, USA, 1:100), mouse monoclonal anti-SAFB (Proteintech, 1:100), rabbit polyclonal anti-SAFB (Proteintech, 1:100), mouse-anti-HSF1 (Abcam, 1:100), rabbit polyclonal anti-heat shock factor 1 (HSF1; Proteintech, 1:100), mouse monoclonal anti-SC35 (Abcam, 1:300), mouse monoclonal anti-coilin (Abcam, 1:200), mouse monoclonal anti-SMN (Sigma, 1:300), mouse monoclonal anti-G3BP (Genentech, 1:100), mouse monoclonal anti-TDP-43 (Proteintech, 1:100), mouse monoclonal anti-FUS (Proteintech, 1:100), and mouse monoclonal anti-HA (Sigma, 1:1,000).

Techniques:

Journal: bioRxiv

Article Title: RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

doi: 10.1101/856880

Figure Lengend Snippet: Assessment of the Association of TDP-43 and FUS with NSBs. (A) HEK293 cells were heat-shocked for 2 hours at 42°C to induce formation of NSBS. Cells were stained for RBM45 and TDP43 or FUS as indicated. Following heat shock, numerous RBM45-positive NSBs become visible in the cell nucleus (arrows) and these do not contain TDP-43 or FUS. Some TDP-43-positive stress granules are visible in the cytoplasm following heat shock and these do not contain RBM45. (B) HEK293 cells were transiently transfected to overexpress HA-tagged TDP-43 or FUS and were then stained for the HA tag and the NSB marker HSF1. Overexpression of TDP-43 was sufficient to robustly induce NSB formation, but NSBs were negative for TDP-43. Neither overexpression of FUS or transfection with a control HA vector resulted in NSB formation. For all images, scale bar = 10 μm.

Article Snippet: The following primary antibodies and dilutions were used for immunofluorescence and immunohistochemistry: rabbit polyclonal anti-RBM45 (Sigma, 1:75), rabbit polyclonal anti-RBM45 (custom; Pacific Immunology, 1:100), mouse monoclonal anti-scaffold attachment factor B (SAFB, Lifespan Biosciences, Seattle, WA, USA, 1:100), mouse monoclonal anti-SAFB (Proteintech, 1:100), rabbit polyclonal anti-SAFB (Proteintech, 1:100), mouse-anti-HSF1 (Abcam, 1:100), rabbit polyclonal anti-heat shock factor 1 (HSF1; Proteintech, 1:100), mouse monoclonal anti-SC35 (Abcam, 1:300), mouse monoclonal anti-coilin (Abcam, 1:200), mouse monoclonal anti-SMN (Sigma, 1:300), mouse monoclonal anti-G3BP (Genentech, 1:100), mouse monoclonal anti-TDP-43 (Proteintech, 1:100), mouse monoclonal anti-FUS (Proteintech, 1:100), and mouse monoclonal anti-HA (Sigma, 1:1,000).

Techniques: Staining, Transfection, Marker, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

doi: 10.1101/856880

Figure Lengend Snippet: Effect of siRNA knockdown of RBM45 and SatIII on NSB formation. HEK293 cells were transfected with siRNAs targeting RBM45, SatIII, or off-target scrambled siRNAs (control). Nuclear stress body (NSB) formation was then assessed by immunocytochemistry. Cells were treated with 1 mM sodium arsenite for 1 hour to induce NSB formation. (A) Effect of off-target, scrambled siRNA (control) on NSB formation. Cells transfected with control siRNAs readily form SAFB, RBM45, and HSF1-positive nuclear stress bodies following treatment with sodium arsenite. (B) Cells transfected with siRNAs targeting RBM45 show reduced levels of RBM45, but readily form SAFB and HSF1-positive NSBs following cellular stress. (C) Effect of SatIII knockdown on NSB formation. Knockdown of SatIII leads to a loss of NSB formation during cellular stress as indicated by the loss of SAFB, RBM45, and HSF1-positive NSBs in cells transfected with SatIII targeting siRNAs. For all images, scale bar = 5 μm.

Article Snippet: The following primary antibodies and dilutions were used for immunofluorescence and immunohistochemistry: rabbit polyclonal anti-RBM45 (Sigma, 1:75), rabbit polyclonal anti-RBM45 (custom; Pacific Immunology, 1:100), mouse monoclonal anti-scaffold attachment factor B (SAFB, Lifespan Biosciences, Seattle, WA, USA, 1:100), mouse monoclonal anti-SAFB (Proteintech, 1:100), rabbit polyclonal anti-SAFB (Proteintech, 1:100), mouse-anti-HSF1 (Abcam, 1:100), rabbit polyclonal anti-heat shock factor 1 (HSF1; Proteintech, 1:100), mouse monoclonal anti-SC35 (Abcam, 1:300), mouse monoclonal anti-coilin (Abcam, 1:200), mouse monoclonal anti-SMN (Sigma, 1:300), mouse monoclonal anti-G3BP (Genentech, 1:100), mouse monoclonal anti-TDP-43 (Proteintech, 1:100), mouse monoclonal anti-FUS (Proteintech, 1:100), and mouse monoclonal anti-HA (Sigma, 1:1,000).

Techniques: Transfection, Immunocytochemistry

Journal: bioRxiv

Article Title: RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

doi: 10.1101/856880

Figure Lengend Snippet: Mapping the RBM45 Domains Required for NSB Incorporation. (A) Schematic showing functional domains and their position in the full-length RBM45 protein. RRM = RNA recognition motif, HOA = homo-oligomerization domain, NLS = nuclear localization sequence. HEK293 cells were transfected with constructs encoding HA-tagged wild-type (WT) or domain-modified forms of RBM45 as indicated to determine which domains of the protein are necessary for incorporation into NSBs. (B) In unstressed cells, the distribution of WT RBM45 is predominately diffuse and nuclear. (C) Heat shock (42°C for 2 hours) leads to the robust formation of NSBs that are positive for RBM45 and the NSB marker HSF1. (D) Removal of the RBM45 NLS leads to its sequestration in the cytoplasm and prevents its association with NSBs during heat shock. (E) Removal of RRM1 does not alter the association of RBM45 with NSBs. (F) Removal of RRM2 prevents the association of RBM45 with NSBs. (G) Removal of RRM3 prevents the association of RBM45 with NSBs. (H) Removal of the RBM45 homo-oligomerization domain (HOA) does not alter the association of RBM45 with NSBs. For all images, scale bar = 5 μm.

Article Snippet: The following primary antibodies and dilutions were used for immunofluorescence and immunohistochemistry: rabbit polyclonal anti-RBM45 (Sigma, 1:75), rabbit polyclonal anti-RBM45 (custom; Pacific Immunology, 1:100), mouse monoclonal anti-scaffold attachment factor B (SAFB, Lifespan Biosciences, Seattle, WA, USA, 1:100), mouse monoclonal anti-SAFB (Proteintech, 1:100), rabbit polyclonal anti-SAFB (Proteintech, 1:100), mouse-anti-HSF1 (Abcam, 1:100), rabbit polyclonal anti-heat shock factor 1 (HSF1; Proteintech, 1:100), mouse monoclonal anti-SC35 (Abcam, 1:300), mouse monoclonal anti-coilin (Abcam, 1:200), mouse monoclonal anti-SMN (Sigma, 1:300), mouse monoclonal anti-G3BP (Genentech, 1:100), mouse monoclonal anti-TDP-43 (Proteintech, 1:100), mouse monoclonal anti-FUS (Proteintech, 1:100), and mouse monoclonal anti-HA (Sigma, 1:1,000).

Techniques: Functional Assay, Sequencing, Transfection, Construct, Modification, Marker

Journal: bioRxiv

Article Title: RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

doi: 10.1101/856880

Figure Lengend Snippet: Chronic Stress Promotes RBM45 Nuclear Inclusion Formation. HEK293 cells were treated with three separate cellular stressors for varying durations to examine the effects of chronic and acute cellular stress on the subcellular distribution of RBM45 and nuclear stress body (NSB) formation. (A and B) In untreated cells, the distribution of RBM45 is diffuse and nuclear and no HSF1 (A) or SAFB (B)-positive NSBs are visible. (C) Acute treatment (6 hours) with the genotoxic stressor mitoxantrone (MTX; 5 µM) induces the formation of RBM45- and HSF1-positive NSBs. (D) Chronic treatment with MTX (24 hours; 1 µM) leads to the formation of RBM45 inclusions via NSB formation. At 24 hours, RBM45 nuclear inclusions are no longer positive for the NSB marker HSF1. (E and F) Similar results were obtained with acute (E) and chronic (F) treatment of cells with the oxidative stressor cadmium sulfate (CdSO4; 30 µM [acute] or 5 µM [chronic]) and the NSB marker SAFB. (G and H) Similar results were also obtained for acute and chronic treatment of HEK293 cells with sodium arsenite (Ars; 1 mM [acute; G] or 0.1 mM [chronic; H]). For all images, scale bar = 5 μm.

Article Snippet: The following primary antibodies and dilutions were used for immunofluorescence and immunohistochemistry: rabbit polyclonal anti-RBM45 (Sigma, 1:75), rabbit polyclonal anti-RBM45 (custom; Pacific Immunology, 1:100), mouse monoclonal anti-scaffold attachment factor B (SAFB, Lifespan Biosciences, Seattle, WA, USA, 1:100), mouse monoclonal anti-SAFB (Proteintech, 1:100), rabbit polyclonal anti-SAFB (Proteintech, 1:100), mouse-anti-HSF1 (Abcam, 1:100), rabbit polyclonal anti-heat shock factor 1 (HSF1; Proteintech, 1:100), mouse monoclonal anti-SC35 (Abcam, 1:300), mouse monoclonal anti-coilin (Abcam, 1:200), mouse monoclonal anti-SMN (Sigma, 1:300), mouse monoclonal anti-G3BP (Genentech, 1:100), mouse monoclonal anti-TDP-43 (Proteintech, 1:100), mouse monoclonal anti-FUS (Proteintech, 1:100), and mouse monoclonal anti-HA (Sigma, 1:1,000).

Techniques: Marker

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: qRT-PCR primers sequences

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques:

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: Aberrantly increased expression of FABP5 in GCs of patients and ovaries of mice with PCOS. A Bar plot showing FABP5 mRNA expression in the human primary ovarian GCs of PCOS patients and healthy individuals, as measured by qRT‒PCR ( n = 12). B Immunofluorescence staining of FABP5 in human primary ovarian GCs of PCOS patients and healthy individuals. Scale bar, 50 μm. C Bar plot showing the expression of Fabp5 mRNA in the ovaries of mice with PCOS measured by qRT‒PCR ( n = 6). D , E Immunoblot plot ( D ) and bar plot of the statistical analysis ( E ) showing the expression of FABP5 in the ovaries of mice with PCOS determined by Western blot analysis ( n = 4). F Immunohistochemical plot of FABP5 expression and localization in the ovaries of mice with PCOS. A two-tailed unpaired t test was used for all the statistical analyses in this section. ** P < 0.01

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Immunohistochemical staining, Two Tailed Test

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: Overexpression of FABP5 promotes fatty acid synthesis in KGN cells. A Bar plot showing the expression of FABP5 in KGN cells after transfection with the PCS2-myc- FABP5 or empty vector plasmids for 48 h, as determined via qRT‒PCR. B , C Immunoblotting plot ( B ) and bar plot of statistical analysis ( C ) showing the expression of FABP5 in KGN cells transfected with PCS2-myc- FABP5 or empty vector plasmids for 48 h. D Nile red staining of KGN cells after 48 h of FABP5 overexpression. The red signal indicates the formation of lipid droplets. All the samples were also stained with DAPI. Scale bar, 100 μm. E Statistical analysis of the number of lipid droplets. Five fields of view were randomly selected. F Bar plot showing the ACSL1 , GPAM1 , LPIN1 and DGAT2 mRNA expression in KGN cells after 48 h of FABP5 expression. A two-tailed unpaired t test was used for all the statistical analyses in this section. * P < 0.05, ** P < 0.01, *** P < 0.001. The groups transfected with the empty vector control or pCS2-myc- FABP5 plasmids were referred to as the EV-ctrl and myc- FABP5 groups, respectively

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Western Blot, Staining, Two Tailed Test, Control

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: Knockdown of FABP5 inhibits fatty acid synthesis in KGN cells. A Bar plot showing the expression of FABP5 in KGN cells after transfection with FABP5 or negative control siRNAs for 48 h. B , C Immunoblot plot ( B ) and bar plot of the statistical analysis ( C ) showing the expression of FABP5 in KGN cells transfected with FABP5 or negative control siRNAs for 48 h. D Nile red staining diagram of KGN cells after transfection with FABP5 siRNA for 48 h. The red signal indicates the formation of lipid droplets. All the samples were also stained with DAPI. Scale bar, 100 μm. E Statistical analysis of the number of lipid droplets. Five fields of view were randomly selected. F Bar plot showing ACSL1 , GPAM1 , LPIN1 and DGAT2 mRNA expression in KGN cells after transfection with FABP5 siRNA for 48 days. A two-tailed unpaired t test was used for all the statistical analyses in this section. * P < 0.05, ** P < 0.01, *** P < 0.001. The groups transfected with the negative control siRNA or with FABP5 siRNA were referred to as siNC and si FABP5 , respectively

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques: Knockdown, Expressing, Transfection, Negative Control, Western Blot, Staining, Two Tailed Test

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: FABP5 facilitates KGN cell proliferation. A Immunoblotting plot showing the expression of FABP5 and the cell proliferation marker PCNA in KGN cells after 48 h of FABP5 overexpression. B A line chart showing the proliferation of KGN cells after 48 h of FABP5 overexpression, as determined by CCK8 assays. C Immunofluorescence staining of Ki67 in KGN cells after 48 h of FABP5 overexpression. All the samples were also stained with DAPI. Scale bar, 100 μm. D Statistical analysis of the Ki67-positive cells. Five fields of view were randomly selected. E Immunoblotting plot showing the expression of FABP5 and PCNA in KGN cells after transfection with FABP5 or negative control siRNAs for 48 h. F Line chart showing the proliferation of KGN cells after transfection with FABP5 or negative control siRNAs for 48 h, as determined by CCK8 assays. G Immunofluorescence staining of Ki67 in KGN cells after transfection with FABP5 or negative control siRNAs for 48 h. All samples were also stained with DAPI. Scale bar, 100 μm. H Statistical analysis of the Ki67-positive cells. Five fields of view were randomly selected. A two-tailed unpaired t test was used for all the statistical analyses. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques: Western Blot, Expressing, Marker, Over Expression, Immunofluorescence, Staining, Transfection, Negative Control, Two Tailed Test

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: FABP5 accelerates the proliferation of KGN cells by activating PI3K-AKT signaling. A Immunoblotting plot showing the expression of FABP5 and AKT and the phosphorylation of AKT in KGN cells after 48 h of FABP5 overexpression or knockdown. B , C Line chart showing the proliferation of KGN cells treated with 10 μM LY294002 or SC79 after 48 h of FABP5 overexpression or knockdown, as determined by CCK8 assays. * represents the comparison between the EV-ctrl and myc- FABP5 or siNC and siFABP5 groups; # represents the comparison between the myc- FABP5 and myc- FABP5 + LY294002 or si FABP5 and si FABP5 + SC79 groups; D , F Immunofluorescence staining of Ki67 in KGN cells treated with 10 μM LY294002 or SC79 for 48 h after FABP5 was overexpressed or knocked down. Scale bar, 100 μm. E , G Statistical analysis of Ki67-positive cells. Five fields of view were randomly selected. * P < 0.05, ** P < 0.01, *** P < 0.001. ## P < 0.01, ### P < 0.01

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques: Western Blot, Expressing, Phospho-proteomics, Over Expression, Knockdown, Comparison, Immunofluorescence, Staining

Journal: Journal of Ovarian Research

Article Title: High levels of fatty acid-binding protein 5 excessively enhances fatty acid synthesis and proliferation of granulosa cells in polycystic ovary syndrome

doi: 10.1186/s13048-024-01368-6

Figure Lengend Snippet: FABP5 accelerates fatty acid synthesis in KGN cells by activating PI3K-AKT signaling ( A , C ) Nile red staining of KGN cells treated with 10 μM LY294002 or SC79 for 48 h after 48 h of FABP5 overexpression or knockdown. The red signal indicates the formation of lipid droplets. All the samples were also stained with DAPI. Scale bar, 100 μm. B , D Statistical analysis of the number of lipid droplets. Five fields of view were randomly selected. E , G Bar plot showing ACSL1 , GPAM1 , LPIN1 and DGAT2 mRNA expression in KGN cells treated with 10 μM LY294002 or SC79 for 48 h after FABP5 overexpression or knockdown. A two-tailed unpaired t test was used for all the statistical analyses. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Subsequently, the KGN cells were incubated overnight with the primary antibody Ki67 (1:2000; Abcam, Shanghai, China) or FABP5 (1:500; Proteintech, Chicago, USA) at 4 °C.

Techniques: Staining, Over Expression, Knockdown, Expressing, Two Tailed Test

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D is upregulated in osteosarcoma tissues and cell lines. a , b , c EEF1D mRNA and protein expression levels in osteosarcoma cell lines (MNNG/HOS, MG63 and U2OS) and human normal osteoblast cell line (hFOB 1.19) were measured by qRT-PCR and western blotting, respectively. d , e EEF1D expression levels were measured in 20 pairs of osteosarcoma and corresponding adjacent non-tumor tissues. For qRT-PCR, EEF1D expression was normalized to β-actin

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D Knockdown inhibits osteosarcoma cell growth in vitro. a - f Expression levels of EEF1D mRNA and protein in MNNG/HOS, U2OS and MG63 cells were measured after EEF1D siRNA (si-EEF1D) transfection by qRT-PCR and western blotting, respectively. g - i Cell Counting Kit-8 (CCK-8) assays were performed to measure cell proliferation after siRNA transfection. j - o Colony-formation assays were performed for EEF1D-silenced osteosarcoma and control cells. Data are representative of results from three independent experiments. * P < 0.05. For qRT-PCR, EEF1D expression was normalized to β-actin

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Knockdown, In Vitro, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Cell Counting, CCK-8 Assay, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D Knockdown inhibits osteosarcoma cell cycle G2/M transition. Representative images of the cell cycle assays in MNNG/HOS ( a , b ), U2OS ( d , e ) and MG63 cells ( g , h ) after transfection with nonspecific control siRNA (si-NC) or EEF1D siRNA (si-EEF1D). c , f , i Diagrams showing the results of cell cycle assay in MNNG/HOS, U2OS and MG63 cells

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Knockdown, Transfection, Control, Cell Cycle Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: EEF1D Knockdown inhibits Akt-mTOR and Akt-Bad signaling pathways in osteosarcoma cells. a , b MNNG/HOS and U2OS cell extracts were prepared and analyzed using PathScan® intracellular signaling array kit. Images were captured with Odyssey® Infrared Imaging System (LI-COR). c , d Quantification of results from MNNG/HOS and U2OS cells shown in ( a , b ) e , f Western blotting analysis of Akt-mTOR and Akt-Bad signaling pathway molecules in MNNG/HOS and U2OS cells transfected with nonspecific control siRNA (si-NC) or EEF1D siRNA (si-EEF1D). g Western blotting analysis of Akt-mTOR and Akt-Bad signaling pathway molecules, including mTOR, Akt, and Bad, in hFOB 1.19 cells transfected with pcDNA 3.1 or pcDNA 3.1-EEF1D. Quantification of results in ( e , f , g ) were shown in ( h , i and j ) respectively

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Knockdown, Protein-Protein interactions, Imaging, Western Blot, Transfection, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: Clinical significance of EEF1D in osteosarcoma patients. a Representative IHC image of EEF1D in non-tumor tissues. b Representative IHC image of EEF1D in osteosarcoma. Original magnification: 50×, 200×

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: EEF1D overexpression promotes osteosarcoma cell proliferation by facilitating Akt-mTOR and Akt-bad signaling

doi: 10.1186/s13046-018-0715-5

Figure Lengend Snippet: Correlation analyses of EEF1D protein expression in relation to clinicopathologic variables of 50 patients with osteosarcoma

Article Snippet: After blocking in 5% nonfat milk, the membranes were incubated with the following primary antibodies: EEF1D (1:500, Proteintech) [ ], mTOR (total, 1:1000; Cell Signaling Technology), phospho-mTOR (Ser2448, 1:1000; Cell Signaling Technology), Akt (total, 1:1000; Cell Signaling Technology), phospho-Akt (Thr308, 1:1000; Cell Signaling Technology), Bad (total, 1:500; BBI Life Sciences), phospho-Bad (Ser112, 1:500; BBI Life Sciences), or β-actin (1:20,000, Sigma-Aldrich).

Techniques: Expressing