mouse gapdh monoclonal  (Proteintech)

 
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    Name:
    Mouse GAPDH Monoclonal
    Description:
    The GAPDH antibody from Proteintech is a mouse monoclonal antibody to a recombinant protein of human GAPDH This antibody recognizes human mouse rat zebrafish yeast plant antigen The GAPDH antibody has been validated for the following applications ELISA FC IF IHC IP WB analysis
    Catalog Number:
    60004-1-Ig
    Price:
    [219.03]
    Applications:
    IHC,Western Blot,ELISA,Flow Cytometry,Immunoprecipitation,Immunofluorescence
    Host:
    Mouse
    Conjugate:
    Unconjugated
    Immunogen:
    Recombinant Protein
    Size:
    150 ul
    Category:
    Antibody
    Antibody Type:
    Primary antibody
    Isotype:
    IgG2b
    Reactivity:
    Human Mouse Pig Rat Zebrafish Yeast Monkey Xenopus Hamster Plant
    Buy from Supplier


    Structured Review

    Proteintech mouse gapdh monoclonal
    miR-137 represses cholangiocarcinoma cell proliferation in vitro . (A) Fluorescence microscope examination (magnification, ×200) and reverse transcription-quantitative PCR were used to detect the infection efficiency of miR-137 overexpression (LV-miR-137) and NC lentiviruses. (B) The effect of miR-137 on cholangiocarcinoma cell proliferation was detected by the Cell Counting Kit-8 assay. (C) The effect of miR-137 on cholangiocarcinoma cell colony formation ability was detected using colony formation assays. (D) Cell cycle distribution was analyzed after miR-137 overexpression in TFK-1 and HuCCT1 cells. (E) Western blotting was used to detect the expression of <t>CDK2</t> and cyclin D1 in the miR-137 overexpression and normal control groups. <t>GAPDH</t> was used as the loading control. * P
    The GAPDH antibody from Proteintech is a mouse monoclonal antibody to a recombinant protein of human GAPDH This antibody recognizes human mouse rat zebrafish yeast plant antigen The GAPDH antibody has been validated for the following applications ELISA FC IF IHC IP WB analysis
    https://www.bioz.com/result/mouse gapdh monoclonal/product/Proteintech
    Average 99 stars, based on 269 article reviews
    Price from $9.99 to $1999.99
    mouse gapdh monoclonal - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "MicroRNA-137 suppresses the proliferation, migration and invasion of cholangiocarcinoma cells by targeting WNT2B"

    Article Title: MicroRNA-137 suppresses the proliferation, migration and invasion of cholangiocarcinoma cells by targeting WNT2B

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2020.4474

    miR-137 represses cholangiocarcinoma cell proliferation in vitro . (A) Fluorescence microscope examination (magnification, ×200) and reverse transcription-quantitative PCR were used to detect the infection efficiency of miR-137 overexpression (LV-miR-137) and NC lentiviruses. (B) The effect of miR-137 on cholangiocarcinoma cell proliferation was detected by the Cell Counting Kit-8 assay. (C) The effect of miR-137 on cholangiocarcinoma cell colony formation ability was detected using colony formation assays. (D) Cell cycle distribution was analyzed after miR-137 overexpression in TFK-1 and HuCCT1 cells. (E) Western blotting was used to detect the expression of CDK2 and cyclin D1 in the miR-137 overexpression and normal control groups. GAPDH was used as the loading control. * P
    Figure Legend Snippet: miR-137 represses cholangiocarcinoma cell proliferation in vitro . (A) Fluorescence microscope examination (magnification, ×200) and reverse transcription-quantitative PCR were used to detect the infection efficiency of miR-137 overexpression (LV-miR-137) and NC lentiviruses. (B) The effect of miR-137 on cholangiocarcinoma cell proliferation was detected by the Cell Counting Kit-8 assay. (C) The effect of miR-137 on cholangiocarcinoma cell colony formation ability was detected using colony formation assays. (D) Cell cycle distribution was analyzed after miR-137 overexpression in TFK-1 and HuCCT1 cells. (E) Western blotting was used to detect the expression of CDK2 and cyclin D1 in the miR-137 overexpression and normal control groups. GAPDH was used as the loading control. * P

    Techniques Used: In Vitro, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Infection, Over Expression, Cell Counting, Western Blot, Expressing

    2) Product Images from "Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia"

    Article Title: Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.225797

    PALD1 accumulates in primary cilia of selected cell types upon Hh pathway activation. (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except for for PALD1 −/− where n = 1). Mean values are indicated by horizontal lines. (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation whereas SMO does. 3T3 cells expressing YFP SMO ( Rohatgi et al., 2009 ) were serum-starved and treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 μm. (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1 −/− cells (see Fig. S4 ). (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) ( Li et al., 2014 ). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors (modified from ( Carvalho-Santos et al., 2010 )). Branch color code: purple, opisthokonts; blue, amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, excavates. When present in the respective organism, motile cilia are shown in green and primary cilia in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs are found (black, 100%; dark grey
    Figure Legend Snippet: PALD1 accumulates in primary cilia of selected cell types upon Hh pathway activation. (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except for for PALD1 −/− where n = 1). Mean values are indicated by horizontal lines. (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation whereas SMO does. 3T3 cells expressing YFP SMO ( Rohatgi et al., 2009 ) were serum-starved and treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 μm. (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1 −/− cells (see Fig. S4 ). (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) ( Li et al., 2014 ). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors (modified from ( Carvalho-Santos et al., 2010 )). Branch color code: purple, opisthokonts; blue, amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, excavates. When present in the respective organism, motile cilia are shown in green and primary cilia in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs are found (black, 100%; dark grey

    Techniques Used: Activation Assay, SDS Page, Western Blot, Expressing, Immunofluorescence, Microscopy, Fluorescence, Mutagenesis, Modification

    3) Product Images from "Protective Effects of Leukemia Inhibitory Factor on Retinal Vasculature and Cells in Streptozotocin-induced Diabetic Mice"

    Article Title: Protective Effects of Leukemia Inhibitory Factor on Retinal Vasculature and Cells in Streptozotocin-induced Diabetic Mice

    Journal: Chinese Medical Journal

    doi: 10.4103/0366-6999.221263

    LIF acts via phosphorylation of STAT3 (p-STAT3). (a) Histogram of the relative protein level of p-STAT3/GAPDH shows that the value of P-STAT3/GAPDH was statistically increased in the LIF-treated group ( n = 4), compared with the diabetic control group ( t = 3.85, P = 0.019, n = 4) and the normal control group ( t = −3.20, P = 0.019, n = 4). It is significant among the groups ( F = 6.40, P = 0.018). * P
    Figure Legend Snippet: LIF acts via phosphorylation of STAT3 (p-STAT3). (a) Histogram of the relative protein level of p-STAT3/GAPDH shows that the value of P-STAT3/GAPDH was statistically increased in the LIF-treated group ( n = 4), compared with the diabetic control group ( t = 3.85, P = 0.019, n = 4) and the normal control group ( t = −3.20, P = 0.019, n = 4). It is significant among the groups ( F = 6.40, P = 0.018). * P

    Techniques Used:

    4) Product Images from "An Efficient Bivalent Cyclic RGD-PIK3CB siRNA Conjugate for Specific Targeted Therapy against Glioblastoma In Vitro and In Vivo"

    Article Title: An Efficient Bivalent Cyclic RGD-PIK3CB siRNA Conjugate for Specific Targeted Therapy against Glioblastoma In Vitro and In Vivo

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.09.002

    Anti-tumor Efficacy and Mechanism of biRGD-siPIK3CB in Orthotopic U87MG-Luc Glioblastoma Model (A) IVIS luminescent imaging of glioblastoma-bearing mice and ex vivo glioblastoma-bearing brain tissues from each group. Conditions of the treatment were as follows: saline group, biRGD-siNC (1 nmol/20 g) group, biRGD-siPIK3CB (0.5 nmol/20 g) group, or biRGD-siPIK3CB (1 nmol/20 g) group. All animals were injected intravenously 12 times over a 24-hr interval. (B) The luminescent signal intensity of mice in all groups. (C and D) Silencing activity of different concentrations of biRGD-siPIK3CB in vivo . U87MG-Luc glioblastoma tissues from the anti-tumor assay were collected for qRT-PCR (C) or western blot (D) analysis; results were normalized with GAPDH level. (E) PIK3CB expression, cell proliferative activity, and the number of apoptotic cells in orthotopic U87MG-Luc glioblastoma tissues were detected by immunohistochemical staining. The relative expression level of PIK3CB protein was evaluated by mean optical density (IOD/area). Proliferative cells in tumor were stained by ki67 antibody, and the results were indicated with ki67-positive cells/total cells. The number of apoptotic cells in tumor was indicated by apoptotic index (TUNEL-positive cells/total cells) with TUNEL staining. Positive cells were showing brown. *p
    Figure Legend Snippet: Anti-tumor Efficacy and Mechanism of biRGD-siPIK3CB in Orthotopic U87MG-Luc Glioblastoma Model (A) IVIS luminescent imaging of glioblastoma-bearing mice and ex vivo glioblastoma-bearing brain tissues from each group. Conditions of the treatment were as follows: saline group, biRGD-siNC (1 nmol/20 g) group, biRGD-siPIK3CB (0.5 nmol/20 g) group, or biRGD-siPIK3CB (1 nmol/20 g) group. All animals were injected intravenously 12 times over a 24-hr interval. (B) The luminescent signal intensity of mice in all groups. (C and D) Silencing activity of different concentrations of biRGD-siPIK3CB in vivo . U87MG-Luc glioblastoma tissues from the anti-tumor assay were collected for qRT-PCR (C) or western blot (D) analysis; results were normalized with GAPDH level. (E) PIK3CB expression, cell proliferative activity, and the number of apoptotic cells in orthotopic U87MG-Luc glioblastoma tissues were detected by immunohistochemical staining. The relative expression level of PIK3CB protein was evaluated by mean optical density (IOD/area). Proliferative cells in tumor were stained by ki67 antibody, and the results were indicated with ki67-positive cells/total cells. The number of apoptotic cells in tumor was indicated by apoptotic index (TUNEL-positive cells/total cells) with TUNEL staining. Positive cells were showing brown. *p

    Techniques Used: Imaging, Mouse Assay, Ex Vivo, Injection, Activity Assay, In Vivo, Quantitative RT-PCR, Western Blot, Expressing, Immunohistochemistry, Staining, TUNEL Assay

    Analyses of Gene-Silencing Efficiency, Cell Cycle, Apoptosis, and Migration of biRGD-siPIK3CB In Vitro Cells were incubated with different molecules for 24 hr in Opti-MEM without serum. Consequently, 10% FBS was added and cells were incubated for a certain amount of time (depending on the assay). (A) Quantitative analysis of PIK3CB mRNA level using qRT-PCR after 48-hr treatment of biRGD-siPIK3CB. The results were normalized with GAPDH mRNA level. (B) Quantitative analysis of PIK3CB protein level using western blot after 72-hr treatment of biRGD-siPIK3CB. The results were normalized with GAPDH protein level. (C) Cell cycle of U87MG cells was analyzed by flow cytometry after 48-hr treatment of biRGD-siPIK3CB. (D) Transwell migration of U87MG cells was analyzed under a microscope after 48-hr treatment of biRGD-siPIK3CB. (E) Apoptosis of U87MG cells was detected by flow cytometry after 72-hr treatment of biRGD-siPIK3CB. *p
    Figure Legend Snippet: Analyses of Gene-Silencing Efficiency, Cell Cycle, Apoptosis, and Migration of biRGD-siPIK3CB In Vitro Cells were incubated with different molecules for 24 hr in Opti-MEM without serum. Consequently, 10% FBS was added and cells were incubated for a certain amount of time (depending on the assay). (A) Quantitative analysis of PIK3CB mRNA level using qRT-PCR after 48-hr treatment of biRGD-siPIK3CB. The results were normalized with GAPDH mRNA level. (B) Quantitative analysis of PIK3CB protein level using western blot after 72-hr treatment of biRGD-siPIK3CB. The results were normalized with GAPDH protein level. (C) Cell cycle of U87MG cells was analyzed by flow cytometry after 48-hr treatment of biRGD-siPIK3CB. (D) Transwell migration of U87MG cells was analyzed under a microscope after 48-hr treatment of biRGD-siPIK3CB. (E) Apoptosis of U87MG cells was detected by flow cytometry after 72-hr treatment of biRGD-siPIK3CB. *p

    Techniques Used: Migration, In Vitro, Incubation, Quantitative RT-PCR, Western Blot, Flow Cytometry, Cytometry, Microscopy

    5) Product Images from "Anti-tumoral potential of MDA19 in human osteosarcoma via suppressing PI3K/Akt/mTOR signaling pathway"

    Article Title: Anti-tumoral potential of MDA19 in human osteosarcoma via suppressing PI3K/Akt/mTOR signaling pathway

    Journal: Bioscience Reports

    doi: 10.1042/BSR20181501

    The effect of apoptosis in OS cells administrated with MDA19 was tested by flow cytometry after Annexin V-FITC/PI staining and Western blot assay ( A ) The change of apoptotic rate in U2OS and MG-63 cells after incubation of MDA19. ( B ) Western blot analysis was performed to test a series of antibodies (Bcl-2, Bax, Cleaved Caspase-3, and Gapdh). All values are expressed as mean ± S.D. ** P
    Figure Legend Snippet: The effect of apoptosis in OS cells administrated with MDA19 was tested by flow cytometry after Annexin V-FITC/PI staining and Western blot assay ( A ) The change of apoptotic rate in U2OS and MG-63 cells after incubation of MDA19. ( B ) Western blot analysis was performed to test a series of antibodies (Bcl-2, Bax, Cleaved Caspase-3, and Gapdh). All values are expressed as mean ± S.D. ** P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Western Blot, Incubation

    6) Product Images from "S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer"

    Article Title: S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer

    Journal: Theranostics

    doi: 10.7150/thno.22552

    β-Trcp binds to Mxi1 and promotes the ubiquitination and degradation of Mxi1. A. Mxi1-associated proteins in HEK293T cells identified by tandem affinity purification and mass spectrometry analysis were presented. The recovered peptide numbers for a given protein were listed as indicated. B. Exogenously expressed β-Trcp interacts with Mxi1 and vice versa. HEK293T cells were transfected with indicated constructs. Cell lysates were subjected to pulldown using S protein beads or immunoprecipitation (IP) using anti-Myc beads and then analyzed by Western blotting using indicated antibodies (n=3). C. Endogenous β-Trcp binds to Mxi1 and vice versa. HeLa cell lysates were subjected to immunopreciptation using IgG or anti-β-Trcp antibodies and then analyzed by Western blotting as indicated (n=3). D. β-Trcp knockdown leads to accumulation of endogenous Mxi1. HeLa and H1299 cells were transfected with control or β-Trcp siRNA for 48 h and then analyzed by Western blotting as indicated (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). E. Overexpression of β-Trcp1 decreases the levels of Mxi1 protein. HeLa cells were transfected with increasing amounts of β-Trcp1, followed by Western blotting with indicated antibodies (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). F. HeLa cells were transfected with indicated constructs for 24 h and treated with MG132 (10 μM) for another 4 h. The samples were subjected to immunopreciptation (IP) using anti-Flag beads and then analyzed by Western blotting using indicated antibodies (n=3). WCL: whole cell lysate. G. Upper panel: HeLa cells transfected with indicated siRNA for 48 h were treated with CHX (20 mg/mL) for the indicated times and then analyzed by Western blotting using indicated antibodies. Lower panel: Quantification of the Mxi1 band intensities over time (n=3). H. Alignment of the candidate phosphodegron sequence in Mxi1 from different species. I. HeLa cells stably expressing empty vector (EV), wild-type Mxi1, or Mxi1 S160A mutant were transfected with indicated constructs and then treated with MG132 (10 μM) for another 4 h. The samples were analyzed as described in (E) (n=3).
    Figure Legend Snippet: β-Trcp binds to Mxi1 and promotes the ubiquitination and degradation of Mxi1. A. Mxi1-associated proteins in HEK293T cells identified by tandem affinity purification and mass spectrometry analysis were presented. The recovered peptide numbers for a given protein were listed as indicated. B. Exogenously expressed β-Trcp interacts with Mxi1 and vice versa. HEK293T cells were transfected with indicated constructs. Cell lysates were subjected to pulldown using S protein beads or immunoprecipitation (IP) using anti-Myc beads and then analyzed by Western blotting using indicated antibodies (n=3). C. Endogenous β-Trcp binds to Mxi1 and vice versa. HeLa cell lysates were subjected to immunopreciptation using IgG or anti-β-Trcp antibodies and then analyzed by Western blotting as indicated (n=3). D. β-Trcp knockdown leads to accumulation of endogenous Mxi1. HeLa and H1299 cells were transfected with control or β-Trcp siRNA for 48 h and then analyzed by Western blotting as indicated (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). E. Overexpression of β-Trcp1 decreases the levels of Mxi1 protein. HeLa cells were transfected with increasing amounts of β-Trcp1, followed by Western blotting with indicated antibodies (n=3). The ratio shows relative Mxi1 protein expression normalized for GAPDH (control, set at 1). F. HeLa cells were transfected with indicated constructs for 24 h and treated with MG132 (10 μM) for another 4 h. The samples were subjected to immunopreciptation (IP) using anti-Flag beads and then analyzed by Western blotting using indicated antibodies (n=3). WCL: whole cell lysate. G. Upper panel: HeLa cells transfected with indicated siRNA for 48 h were treated with CHX (20 mg/mL) for the indicated times and then analyzed by Western blotting using indicated antibodies. Lower panel: Quantification of the Mxi1 band intensities over time (n=3). H. Alignment of the candidate phosphodegron sequence in Mxi1 from different species. I. HeLa cells stably expressing empty vector (EV), wild-type Mxi1, or Mxi1 S160A mutant were transfected with indicated constructs and then treated with MG132 (10 μM) for another 4 h. The samples were analyzed as described in (E) (n=3).

    Techniques Used: Affinity Purification, Mass Spectrometry, Transfection, Construct, Immunoprecipitation, Western Blot, Expressing, Over Expression, Sequencing, Stable Transfection, Plasmid Preparation, Mutagenesis

    7) Product Images from "BET bromodomain inhibitor JQ1 preferentially suppresses EBV-positive nasopharyngeal carcinoma cells partially through repressing c-Myc"

    Article Title: BET bromodomain inhibitor JQ1 preferentially suppresses EBV-positive nasopharyngeal carcinoma cells partially through repressing c-Myc

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0789-1

    JQ1 represses TP63, TP53 and their targets, and PD-L1 in NPC. a Venn diagram showing the overlap of significantly altered genes after exposure to 1 μM JQ1 for 3 h in CNE2-EBV−/+ and TWO3-EBV−/+ cell lines. b Heatmap representation of the top-30 down- and up-regulated genes following 3 h of 1 μM JQ1 treatment of all CNE2-EBV−/+ and TWO3-EBV−/+ cell lines. Data are presented as row normalized. c GSEA showing downregulation of TP63-dependent gene sets in the transcriptional profiles of NPC cell lines treated with JQ1. d GSEA displaying downregulation of TP53- and TP63-dependent gene sets in the transcriptional profiles of NPC cell lines treated with JQ1. e Western blot analysis of antibodies against TP63 of CNE1, CNE2, and CNE2-EBV+ cells after treatment with DMSO, JQ1 (500 nM), IR (4 Gy), or 500 nM JQ1 for 48 h followed by IR of 4 Gy. GAPDH served as a loading control. f CNE2-EBV−/+ and TWO3-EBV−/+ cells were treated with indicated concentrations of JQ1 for 24 h and were analyzed for PD-L1 level by western blot analysis. GAPDH served as a loading control. g CNE2-EBV−/+ and TWO3-EBV−/+ cells were treated with 1 μM JQ1 for 0, 24, 48 and 72 h and analyzed for PD-L1 level by western blot analysis. GAPDH served as a loading control
    Figure Legend Snippet: JQ1 represses TP63, TP53 and their targets, and PD-L1 in NPC. a Venn diagram showing the overlap of significantly altered genes after exposure to 1 μM JQ1 for 3 h in CNE2-EBV−/+ and TWO3-EBV−/+ cell lines. b Heatmap representation of the top-30 down- and up-regulated genes following 3 h of 1 μM JQ1 treatment of all CNE2-EBV−/+ and TWO3-EBV−/+ cell lines. Data are presented as row normalized. c GSEA showing downregulation of TP63-dependent gene sets in the transcriptional profiles of NPC cell lines treated with JQ1. d GSEA displaying downregulation of TP53- and TP63-dependent gene sets in the transcriptional profiles of NPC cell lines treated with JQ1. e Western blot analysis of antibodies against TP63 of CNE1, CNE2, and CNE2-EBV+ cells after treatment with DMSO, JQ1 (500 nM), IR (4 Gy), or 500 nM JQ1 for 48 h followed by IR of 4 Gy. GAPDH served as a loading control. f CNE2-EBV−/+ and TWO3-EBV−/+ cells were treated with indicated concentrations of JQ1 for 24 h and were analyzed for PD-L1 level by western blot analysis. GAPDH served as a loading control. g CNE2-EBV−/+ and TWO3-EBV−/+ cells were treated with 1 μM JQ1 for 0, 24, 48 and 72 h and analyzed for PD-L1 level by western blot analysis. GAPDH served as a loading control

    Techniques Used: Western Blot

    JQ1 induces G2/M cell cycle arrest of NPC cells after irradiation. a Cell cycle analysis of CNE1, CNE2, and CNE2-EBV+ cells after treatment with DMSO, JQ1 (500 nM), IR (4 Gy), or 500 nM JQ1 for 48 h followed by IR of 4 Gy. b Quantification of each cell cycle phase in CNE1, CNE2, and CNE2-EBV+ cells in each group shown in a . c Western blot analysis of antibodies against p21 of CNE1, CNE2, and CNE2-EBV+ cells after treatment with DMSO, JQ1 (500 nM), IR (4 Gy), or 500 nM JQ1 for 48 h followed by IR of 4 Gy. GAPDH served as a loading control. d Western blot analyses of the concentration- and time-dependent effect of JQ1 treatment on p21 expression in CNE2-EBV−/+ cell lines
    Figure Legend Snippet: JQ1 induces G2/M cell cycle arrest of NPC cells after irradiation. a Cell cycle analysis of CNE1, CNE2, and CNE2-EBV+ cells after treatment with DMSO, JQ1 (500 nM), IR (4 Gy), or 500 nM JQ1 for 48 h followed by IR of 4 Gy. b Quantification of each cell cycle phase in CNE1, CNE2, and CNE2-EBV+ cells in each group shown in a . c Western blot analysis of antibodies against p21 of CNE1, CNE2, and CNE2-EBV+ cells after treatment with DMSO, JQ1 (500 nM), IR (4 Gy), or 500 nM JQ1 for 48 h followed by IR of 4 Gy. GAPDH served as a loading control. d Western blot analyses of the concentration- and time-dependent effect of JQ1 treatment on p21 expression in CNE2-EBV−/+ cell lines

    Techniques Used: Irradiation, Cell Cycle Assay, Western Blot, Concentration Assay, Expressing

    8) Product Images from "MicroRNA-145-5p inhibits osteosarcoma cell proliferation by targeting E2F transcription factor 3"

    Article Title: MicroRNA-145-5p inhibits osteosarcoma cell proliferation by targeting E2F transcription factor 3

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2020.4504

    miR-145-5p induces G 1 phase arrest in osteosarcoma cells. (A) Cell cycle distribution analysis in 143B and Well5 cells overexpressing miR-145-5p. (B) Western blotting was used to detect the expression of CDK2, CDK4, CDK6, cyclin E and cyclin D1 in cells overexpressing miR-145-5p. GAPDH was used as loading control. ** P
    Figure Legend Snippet: miR-145-5p induces G 1 phase arrest in osteosarcoma cells. (A) Cell cycle distribution analysis in 143B and Well5 cells overexpressing miR-145-5p. (B) Western blotting was used to detect the expression of CDK2, CDK4, CDK6, cyclin E and cyclin D1 in cells overexpressing miR-145-5p. GAPDH was used as loading control. ** P

    Techniques Used: Western Blot, Expressing

    9) Product Images from "The HMGA2-IMP2 Pathway Promotes Granulosa Cell Proliferation in Polycystic Ovary Syndrome"

    Article Title: The HMGA2-IMP2 Pathway Promotes Granulosa Cell Proliferation in Polycystic Ovary Syndrome

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2018-00544

    CCND2 and SERBP1 expression in the HMGA2-IMP2 pathway. (A, B) IMP2 binding sites in the CCND2 and SERBP1 genes. IMP2 bound to CCND2 and SERBP1 mRNAs. (C) RIP experiments in HEK293 cells. CCND2 and SERBP1 proteins were pulled down using anti-IMP2 antibodies. IgG and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as negative controls. (D) Knockdown of HMGA2 in KGN cells reduced the CCND2 and SERBP1 protein levels. (E) Knockdown of IMP2 reduced the CCND2 and SERBP1 protein levels in KGN cells. (F, G) Luciferase assays of IMP2 and downstream genes (F, CCND2 ; G, SERBP1 ) in KGN cells. In both cases, a dose-dependent increase in luciferase activity of CCND2 and SERBP1 was observed as IMP2 expression increased. * P
    Figure Legend Snippet: CCND2 and SERBP1 expression in the HMGA2-IMP2 pathway. (A, B) IMP2 binding sites in the CCND2 and SERBP1 genes. IMP2 bound to CCND2 and SERBP1 mRNAs. (C) RIP experiments in HEK293 cells. CCND2 and SERBP1 proteins were pulled down using anti-IMP2 antibodies. IgG and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as negative controls. (D) Knockdown of HMGA2 in KGN cells reduced the CCND2 and SERBP1 protein levels. (E) Knockdown of IMP2 reduced the CCND2 and SERBP1 protein levels in KGN cells. (F, G) Luciferase assays of IMP2 and downstream genes (F, CCND2 ; G, SERBP1 ) in KGN cells. In both cases, a dose-dependent increase in luciferase activity of CCND2 and SERBP1 was observed as IMP2 expression increased. * P

    Techniques Used: Expressing, Binding Assay, Luciferase, Activity Assay

    CCND2 and SERBP1 mRNA and protein expression in control and PCOS GCs. (A, B) CCND2 mRNA and protein expression was higher in PCOS GCs than in control cells. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ** P
    Figure Legend Snippet: CCND2 and SERBP1 mRNA and protein expression in control and PCOS GCs. (A, B) CCND2 mRNA and protein expression was higher in PCOS GCs than in control cells. Data were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ** P

    Techniques Used: Expressing

    10) Product Images from "Bioinformatics Analysis Identifies p53 as a Candidate Prognostic Biomarker for Neuropathic Pain"

    Article Title: Bioinformatics Analysis Identifies p53 as a Candidate Prognostic Biomarker for Neuropathic Pain

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2018.00320

    Chronic constriction injury (CCI)-induced hyperalgesia and expression of p53 and caspase-3 in dorsal root ganglion (DRG) neurons. (A) Compared to the sham group mice, the CCI mice had decreased paw withdrawal latency (PWL) from day 3 after surgery. (B) Western blot analysis of the expression of p53 and caspase-3 on day 7 after CCI or sham surgery. The fold change of p53 and caspase-3 was normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level. (C) Quantification of p53 and caspase-3 expression. Double immunofluorescence staining for p53 and caspase-3 in DRG neurons. (D) Representative immunofluorescence staining of p53 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (E) Representative immunofluorescence staining of caspase-3 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (F,G) Quantitative analysis of p53-positive and caspase-3-positive cells (%) in DRG tissues at 7 days after CCI. (H) Median fluorescence of NeuN. Magnification: 100× for all columns. Data are all expressed as mean ± SD. ∗ P
    Figure Legend Snippet: Chronic constriction injury (CCI)-induced hyperalgesia and expression of p53 and caspase-3 in dorsal root ganglion (DRG) neurons. (A) Compared to the sham group mice, the CCI mice had decreased paw withdrawal latency (PWL) from day 3 after surgery. (B) Western blot analysis of the expression of p53 and caspase-3 on day 7 after CCI or sham surgery. The fold change of p53 and caspase-3 was normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level. (C) Quantification of p53 and caspase-3 expression. Double immunofluorescence staining for p53 and caspase-3 in DRG neurons. (D) Representative immunofluorescence staining of p53 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (E) Representative immunofluorescence staining of caspase-3 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (F,G) Quantitative analysis of p53-positive and caspase-3-positive cells (%) in DRG tissues at 7 days after CCI. (H) Median fluorescence of NeuN. Magnification: 100× for all columns. Data are all expressed as mean ± SD. ∗ P

    Techniques Used: Expressing, Mouse Assay, Western Blot, Double Immunofluorescence Staining, Immunofluorescence, Staining, Fluorescence

    Inhibition of p53 by pifithrin-α (HY-15484) restrained the pain behaviors and expression of p53 and caspase-3 in dorsal root ganglion (DRG) neurons. (A) Thermal hyperalgesia after intrathecal injection of pifithrin-α (10 μg). Pifithrin-α attenuated chronic constriction injury (CCI)-induced thermal hyperalgesia, compared with that in the CCI + DMSO group. (B) Western blot analysis of the expression of p53 and caspase-3 after intrathecal administration of pifithrin-α on day 7 after CCI. The fold change of p53 and caspase-3 levels was normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level. (C) Quantification of p53 and caspase-3 expression. Double immunofluorescence staining for p53 and caspase-3 in DRG neurons after intrathecal administration of pifithrin-α on day 7 after CCI. (D) Representative immunofluorescence staining of p53 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (E) Representative immunofluorescence staining of caspase-3 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (F,G) Quantitative analysis of p53-positive and caspase-3-positive cells (%) in DRG tissues at 7 days after intrathecal administration of pifithrin-α in the CCI model. (H) Median fluorescence of NeuN. Magnification: 100× for all columns. Data are all expressed as mean ± SD. # P
    Figure Legend Snippet: Inhibition of p53 by pifithrin-α (HY-15484) restrained the pain behaviors and expression of p53 and caspase-3 in dorsal root ganglion (DRG) neurons. (A) Thermal hyperalgesia after intrathecal injection of pifithrin-α (10 μg). Pifithrin-α attenuated chronic constriction injury (CCI)-induced thermal hyperalgesia, compared with that in the CCI + DMSO group. (B) Western blot analysis of the expression of p53 and caspase-3 after intrathecal administration of pifithrin-α on day 7 after CCI. The fold change of p53 and caspase-3 levels was normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level. (C) Quantification of p53 and caspase-3 expression. Double immunofluorescence staining for p53 and caspase-3 in DRG neurons after intrathecal administration of pifithrin-α on day 7 after CCI. (D) Representative immunofluorescence staining of p53 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (E) Representative immunofluorescence staining of caspase-3 (red) and its colocalization with neurons (NeuN, green) in DRG tissues. (F,G) Quantitative analysis of p53-positive and caspase-3-positive cells (%) in DRG tissues at 7 days after intrathecal administration of pifithrin-α in the CCI model. (H) Median fluorescence of NeuN. Magnification: 100× for all columns. Data are all expressed as mean ± SD. # P

    Techniques Used: Inhibition, Expressing, Injection, Western Blot, Double Immunofluorescence Staining, Immunofluorescence, Staining, Fluorescence

    11) Product Images from "PJA1 Coordinates with the SMC5/6 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner"

    Article Title: PJA1 Coordinates with the SMC5/6 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner

    Journal: Journal of Virology

    doi: 10.1128/JVI.00825-18

    PJA1 generally represses viral and episomal DNAs independent of IFN signaling pathways. (A and B) 293T cells (A) or HepG2 cells (B) were plated in 12-well plates and transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h. The mRNA levels of endogenous IFN -α, IFN -β, and IFN -γ genes were measured by RT-qPCR. (C to E) 293T cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with recombinant human IFN-α (rhIFN-α) (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA levels of endogenous PKR (C), OAS1 (D), and MX1 (E) genes were measured by RT-qPCR. (F) HepG2 cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA level of the endogenous PKR gene was measured by RT-qPCR. (G and H) 293T cells (G) or HepG2 cells (H) were treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The PJA1 and GAPDH mRNA levels were measured by RT-qPCR. (I) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA or pCAGGS-HA-PJA1B and 0.2 μg reporters, pIFN-β-Luc, pISRE-Luc, pNF-κB-Luc, pTp53-Luc, and pCMV-Luc for 24 h. Luciferase activities were measured. (J) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1B and 0.2 μg pCMV-Luc for 24 h. Luciferase activities were measured. (K to M) 293T cells were plated in 24-well plates and cotransfected with 0.2 μg pCMV-Luc and pCAGGS-HA-PJA1B at 0, 0.01, 0.05, and 0.1 μg for 24 h. (K) Luciferase activity was measured. (L) The Luc mRNA level was quantified by RT-qPCR. (M) pCMV-Luc DNA in the nuclear extract was quantified by qPCR. (N to P) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR and 0.2 μg reporters, pISRE-Luc (L), pCMV-Luc (M), and pTK-Renilla-Luc (N) for 24 h. Luciferase activities were measured. (Q and R) 293T cells were plated in 12-well plates and transfected with 0.5 μg pEGFP and 0.5 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR for 24 h. (Q) GFP, PJA1, and β-actin were detected by Western blotting. WCL, whole-cell lysate. (R) Fluorescence intensity of EGFP was detected. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. Results are expressed as fold induction relative to the control. ns, not significant ( P > 0.05); *, P ≤ 0.05; **, P
    Figure Legend Snippet: PJA1 generally represses viral and episomal DNAs independent of IFN signaling pathways. (A and B) 293T cells (A) or HepG2 cells (B) were plated in 12-well plates and transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h. The mRNA levels of endogenous IFN -α, IFN -β, and IFN -γ genes were measured by RT-qPCR. (C to E) 293T cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with recombinant human IFN-α (rhIFN-α) (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA levels of endogenous PKR (C), OAS1 (D), and MX1 (E) genes were measured by RT-qPCR. (F) HepG2 cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA level of the endogenous PKR gene was measured by RT-qPCR. (G and H) 293T cells (G) or HepG2 cells (H) were treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The PJA1 and GAPDH mRNA levels were measured by RT-qPCR. (I) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA or pCAGGS-HA-PJA1B and 0.2 μg reporters, pIFN-β-Luc, pISRE-Luc, pNF-κB-Luc, pTp53-Luc, and pCMV-Luc for 24 h. Luciferase activities were measured. (J) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1B and 0.2 μg pCMV-Luc for 24 h. Luciferase activities were measured. (K to M) 293T cells were plated in 24-well plates and cotransfected with 0.2 μg pCMV-Luc and pCAGGS-HA-PJA1B at 0, 0.01, 0.05, and 0.1 μg for 24 h. (K) Luciferase activity was measured. (L) The Luc mRNA level was quantified by RT-qPCR. (M) pCMV-Luc DNA in the nuclear extract was quantified by qPCR. (N to P) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR and 0.2 μg reporters, pISRE-Luc (L), pCMV-Luc (M), and pTK-Renilla-Luc (N) for 24 h. Luciferase activities were measured. (Q and R) 293T cells were plated in 12-well plates and transfected with 0.5 μg pEGFP and 0.5 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR for 24 h. (Q) GFP, PJA1, and β-actin were detected by Western blotting. WCL, whole-cell lysate. (R) Fluorescence intensity of EGFP was detected. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. Results are expressed as fold induction relative to the control. ns, not significant ( P > 0.05); *, P ≤ 0.05; **, P

    Techniques Used: Transfection, Quantitative RT-PCR, Recombinant, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence

    PJA1 has no effect on chromosome-integrated genes and RNA viruses. (A) 293T-Luc cells were generated, in which the CMV promoter driving Luc was randomly integrated into cell chromosomes by a lentiviral system. The cells were plated in 24-well plates and transfected with 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Luciferase activities were measured. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. ns, nonsignificant. (B) 293T-EGFP cells were generated, in which the CMV promoter driving EGFP was randomly integrated into cellular chromosomes by a lentiviral system. The cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR. GFP and β-actin protein were detected by Western blot analyses. (C) 293T cells were plated in 12-well plates and cotransfected with 0.5 μg pFlag-IRAK1 and 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Flag-IRAK1 and GAPDH protein levels were determined by Western blot analyses. (D) 293T cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Endogenous IRAK1 and GAPDH were detected by Western blot analyses. (E) HepG2 cells were plated in 12-well plates and cotransfected with 0.5 μg pCAGGS-HA-IFNAR1 and 0.5 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. HA-IFNAR1 and GAPDH were detected by Western blot analyses. (F) HepG2 cells were plated in 12-well plates and transfected with 1 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. Endogenous IFNAR1 and GAPDH were detected by Western blot analyses. (G) RD cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 48 h, and infected with EV71 at an MOI of 5 for 6 h. EV71 VP1, HA-PJA1B, and GAPDH were detected by Western blot analyses. (H) 293T cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 24 h, and infected with VSV-GFP at an MOI of 1 for 12 h. GFP, PJA1B, and GAPDH were detected by Western blot analyses.
    Figure Legend Snippet: PJA1 has no effect on chromosome-integrated genes and RNA viruses. (A) 293T-Luc cells were generated, in which the CMV promoter driving Luc was randomly integrated into cell chromosomes by a lentiviral system. The cells were plated in 24-well plates and transfected with 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Luciferase activities were measured. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. ns, nonsignificant. (B) 293T-EGFP cells were generated, in which the CMV promoter driving EGFP was randomly integrated into cellular chromosomes by a lentiviral system. The cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR. GFP and β-actin protein were detected by Western blot analyses. (C) 293T cells were plated in 12-well plates and cotransfected with 0.5 μg pFlag-IRAK1 and 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Flag-IRAK1 and GAPDH protein levels were determined by Western blot analyses. (D) 293T cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Endogenous IRAK1 and GAPDH were detected by Western blot analyses. (E) HepG2 cells were plated in 12-well plates and cotransfected with 0.5 μg pCAGGS-HA-IFNAR1 and 0.5 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. HA-IFNAR1 and GAPDH were detected by Western blot analyses. (F) HepG2 cells were plated in 12-well plates and transfected with 1 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. Endogenous IFNAR1 and GAPDH were detected by Western blot analyses. (G) RD cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 48 h, and infected with EV71 at an MOI of 5 for 6 h. EV71 VP1, HA-PJA1B, and GAPDH were detected by Western blot analyses. (H) 293T cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 24 h, and infected with VSV-GFP at an MOI of 1 for 12 h. GFP, PJA1B, and GAPDH were detected by Western blot analyses.

    Techniques Used: Generated, Transfection, Luciferase, Western Blot, Infection

    12) Product Images from "Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression"

    Article Title: Curcumae Radix Extract Decreases Mammary Tumor-Derived Lung Metastasis via Suppression of C-C Chemokine Receptor Type 7 Expression

    Journal: Nutrients

    doi: 10.3390/nu11020410

    Downregulation of CCR7 gene by siRNA inhibits gene expression of MMP9, c-FOS, c-JUN and CCL21 in vitro. ( A ) Western blot of proteins extracted from MCF7 cell against CCR7 and GAPDH. ( B )To investigate level of transcripts associated with CCR7, MMP9, CCR7, c-FOS, c-JUN and CCL21 were monitored in MCF7 cell transfected with CCR7 siRNA by using qPCR. Β-ACTIN and 18S mRNA was used as an internal control in real time PCR. Data are representative of four independent experiments and are expressed as the mean ± SD; * p
    Figure Legend Snippet: Downregulation of CCR7 gene by siRNA inhibits gene expression of MMP9, c-FOS, c-JUN and CCL21 in vitro. ( A ) Western blot of proteins extracted from MCF7 cell against CCR7 and GAPDH. ( B )To investigate level of transcripts associated with CCR7, MMP9, CCR7, c-FOS, c-JUN and CCL21 were monitored in MCF7 cell transfected with CCR7 siRNA by using qPCR. Β-ACTIN and 18S mRNA was used as an internal control in real time PCR. Data are representative of four independent experiments and are expressed as the mean ± SD; * p

    Techniques Used: Expressing, In Vitro, Western Blot, Transfection, Real-time Polymerase Chain Reaction

    13) Product Images from "Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer"

    Article Title: Long noncoding RNA OCC-1 suppresses cell growth through destabilizing HuR protein in colorectal cancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky214

    OCC-1 RNA associates with HuR protein. ( A ) RNA pull-down assay followed by western blot confirmed HuR as a protein partner binding specifically to OCC-1 3′UTR. EGFP RNA was used as a RNA control. ACTB and GAPDH are protein controls. ( B ) RIP confirmed the association between OCC-1 and HuR in Caco-2 cells. GAPDH mRNA was used as a non-HuR target control. ( C ) Schematic of the 3′UTRs of OCC-1 orthologues showing the HuR-binding motifs. The HuR-binding sites (HuR CLIP site) identified previously by a CLIP experiment in human OCC-1 3′UTR were also indicated. ( D ) Venn diagram demonstrating the significant enrichment of OCC-1-repressed genes in the set of HuR targets determined by the previous CLIP experiment. About 74% of OCC-1-repressed genes identified by microarray analysis were also HuR targets. ( E ) RIP assay revealed that the mRNAs of the six selected OCC-1-repressed genes also interact with HuR in Caco-2 cells. ( F ) The mRNA and protein level of HuR were markedly reduced by a HuR-targeting shRNA as determined by RT-qPCR ( left ) and western blot ( right ). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. ( G ) RT-qPCR analysis revealed that OCC-1 and all six selected OCC-1-repressed genes were downregulated by HuR knockdown. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. ** P
    Figure Legend Snippet: OCC-1 RNA associates with HuR protein. ( A ) RNA pull-down assay followed by western blot confirmed HuR as a protein partner binding specifically to OCC-1 3′UTR. EGFP RNA was used as a RNA control. ACTB and GAPDH are protein controls. ( B ) RIP confirmed the association between OCC-1 and HuR in Caco-2 cells. GAPDH mRNA was used as a non-HuR target control. ( C ) Schematic of the 3′UTRs of OCC-1 orthologues showing the HuR-binding motifs. The HuR-binding sites (HuR CLIP site) identified previously by a CLIP experiment in human OCC-1 3′UTR were also indicated. ( D ) Venn diagram demonstrating the significant enrichment of OCC-1-repressed genes in the set of HuR targets determined by the previous CLIP experiment. About 74% of OCC-1-repressed genes identified by microarray analysis were also HuR targets. ( E ) RIP assay revealed that the mRNAs of the six selected OCC-1-repressed genes also interact with HuR in Caco-2 cells. ( F ) The mRNA and protein level of HuR were markedly reduced by a HuR-targeting shRNA as determined by RT-qPCR ( left ) and western blot ( right ). The density of protein bands was measured by Image J software and the relative level of HuR protein was calculated after normalizing to ACTB protein. ( G ) RT-qPCR analysis revealed that OCC-1 and all six selected OCC-1-repressed genes were downregulated by HuR knockdown. Images are the result of one representative experiment. Data are presented as mean± standard deviation of three independent experiments. ** P

    Techniques Used: Pull Down Assay, Western Blot, Binding Assay, Cross-linking Immunoprecipitation, Microarray, shRNA, Quantitative RT-PCR, Software, Standard Deviation

    14) Product Images from "Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells"

    Article Title: Dengue Virus Infects Primary Human Hair Follicle Dermal Papilla Cells

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00268

    DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P
    Figure Legend Snippet: DENV-1 and−2 induce interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), signal transducer, and activator of transcription 1 (STAT1), and IL-12b gene expression in HFDPCs. RT-qPCR of IL-6, TNF-α, STAT1, and IL-12b expression in HFDPCs infected with DENV-1 (MOI = 10) and DENV-2 (MOI = 10 and 50) for 4 days. The gene expression was normalized to GAPDH gene. Data are mean ± SD from three independent tests, * P

    Techniques Used: Expressing, Quantitative RT-PCR, Infection

    15) Product Images from "Demethylation of the HACE1 gene promoter inhibits the proliferation of human liver cancer cells"

    Article Title: Demethylation of the HACE1 gene promoter inhibits the proliferation of human liver cancer cells

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10139

    Demethylation of HACE1 promotes OPTN ubiquitination and autophagy. sgRNA-mediated HACE1 demethylation promoted OPTN ubiquitination. Hep3B or HepG2 cells were co-transfected with sgHACE1 (1, 4 and 5)-guided dCas9-Tet1-CD and MS2-Tet1-CD for 48 h. Subsequently, cell lysates were immunoprecipitated with an anti-OPTN antibody and subjected to immunoblotting. LC3 II/I expression was also detected. Inputs refers to whole cell lysates, and GAPDH was used as a loading control. Cas, clustered regularly interspaced short palindromic repeats-associated; HACE1, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1; IB, immunoblotting; LC3, microtubule-associated proteins 1A/1B light chain 3B; OPTN, optineurin; sgRNA, single guide RNA; UB, ubiquitin.
    Figure Legend Snippet: Demethylation of HACE1 promotes OPTN ubiquitination and autophagy. sgRNA-mediated HACE1 demethylation promoted OPTN ubiquitination. Hep3B or HepG2 cells were co-transfected with sgHACE1 (1, 4 and 5)-guided dCas9-Tet1-CD and MS2-Tet1-CD for 48 h. Subsequently, cell lysates were immunoprecipitated with an anti-OPTN antibody and subjected to immunoblotting. LC3 II/I expression was also detected. Inputs refers to whole cell lysates, and GAPDH was used as a loading control. Cas, clustered regularly interspaced short palindromic repeats-associated; HACE1, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1; IB, immunoblotting; LC3, microtubule-associated proteins 1A/1B light chain 3B; OPTN, optineurin; sgRNA, single guide RNA; UB, ubiquitin.

    Techniques Used: Transfection, Immunoprecipitation, Expressing, CRISPR

    16) Product Images from "PJA1 Coordinates with the SMC5/6 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner"

    Article Title: PJA1 Coordinates with the SMC5/6 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner

    Journal: Journal of Virology

    doi: 10.1128/JVI.00825-18

    PJA1 generally represses viral and episomal DNAs independent of IFN signaling pathways. (A and B) 293T cells (A) or HepG2 cells (B) were plated in 12-well plates and transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h. The mRNA levels of endogenous IFN -α, IFN -β, and IFN -γ genes were measured by RT-qPCR. (C to E) 293T cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with recombinant human IFN-α (rhIFN-α) (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA levels of endogenous PKR (C), OAS1 (D), and MX1 (E) genes were measured by RT-qPCR. (F) HepG2 cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA level of the endogenous PKR gene was measured by RT-qPCR. (G and H) 293T cells (G) or HepG2 cells (H) were treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The PJA1 and GAPDH mRNA levels were measured by RT-qPCR. (I) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA or pCAGGS-HA-PJA1B and 0.2 μg reporters, pIFN-β-Luc, pISRE-Luc, pNF-κB-Luc, pTp53-Luc, and pCMV-Luc for 24 h. Luciferase activities were measured. (J) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1B and 0.2 μg pCMV-Luc for 24 h. Luciferase activities were measured. (K to M) 293T cells were plated in 24-well plates and cotransfected with 0.2 μg pCMV-Luc and pCAGGS-HA-PJA1B at 0, 0.01, 0.05, and 0.1 μg for 24 h. (K) Luciferase activity was measured. (L) The Luc mRNA level was quantified by RT-qPCR. (M) pCMV-Luc DNA in the nuclear extract was quantified by qPCR. (N to P) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR and 0.2 μg reporters, pISRE-Luc (L), pCMV-Luc (M), and pTK-Renilla-Luc (N) for 24 h. Luciferase activities were measured. (Q and R) 293T cells were plated in 12-well plates and transfected with 0.5 μg pEGFP and 0.5 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR for 24 h. (Q) GFP, PJA1, and β-actin were detected by Western blotting. WCL, whole-cell lysate. (R) Fluorescence intensity of EGFP was detected. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. Results are expressed as fold induction relative to the control. ns, not significant ( P > 0.05); *, P ≤ 0.05; **, P
    Figure Legend Snippet: PJA1 generally represses viral and episomal DNAs independent of IFN signaling pathways. (A and B) 293T cells (A) or HepG2 cells (B) were plated in 12-well plates and transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h. The mRNA levels of endogenous IFN -α, IFN -β, and IFN -γ genes were measured by RT-qPCR. (C to E) 293T cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with recombinant human IFN-α (rhIFN-α) (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA levels of endogenous PKR (C), OAS1 (D), and MX1 (E) genes were measured by RT-qPCR. (F) HepG2 cells were plated in 12-well plates; transfected with 1 μg pCAGGS-HA or pCAGGS-HA-PJA1B for 24 h; and treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The mRNA level of the endogenous PKR gene was measured by RT-qPCR. (G and H) 293T cells (G) or HepG2 cells (H) were treated with rhIFN-α (300 IU/ml), rhIFN-β (10 ng/ml), and rhIFN-γ (50 ng/ml) for 12 h. The PJA1 and GAPDH mRNA levels were measured by RT-qPCR. (I) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA or pCAGGS-HA-PJA1B and 0.2 μg reporters, pIFN-β-Luc, pISRE-Luc, pNF-κB-Luc, pTp53-Luc, and pCMV-Luc for 24 h. Luciferase activities were measured. (J) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1B and 0.2 μg pCMV-Luc for 24 h. Luciferase activities were measured. (K to M) 293T cells were plated in 24-well plates and cotransfected with 0.2 μg pCMV-Luc and pCAGGS-HA-PJA1B at 0, 0.01, 0.05, and 0.1 μg for 24 h. (K) Luciferase activity was measured. (L) The Luc mRNA level was quantified by RT-qPCR. (M) pCMV-Luc DNA in the nuclear extract was quantified by qPCR. (N to P) 293T cells were plated in 24-well plates and cotransfected with 0.3 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR and 0.2 μg reporters, pISRE-Luc (L), pCMV-Luc (M), and pTK-Renilla-Luc (N) for 24 h. Luciferase activities were measured. (Q and R) 293T cells were plated in 12-well plates and transfected with 0.5 μg pEGFP and 0.5 μg pCAGGS-HA, pCAGGS-HA-PJA1B, or pCAGGS-HA-PJA1BΔR for 24 h. (Q) GFP, PJA1, and β-actin were detected by Western blotting. WCL, whole-cell lysate. (R) Fluorescence intensity of EGFP was detected. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. Results are expressed as fold induction relative to the control. ns, not significant ( P > 0.05); *, P ≤ 0.05; **, P

    Techniques Used: Transfection, Quantitative RT-PCR, Recombinant, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence

    PJA1 has no effect on chromosome-integrated genes and RNA viruses. (A) 293T-Luc cells were generated, in which the CMV promoter driving Luc was randomly integrated into cell chromosomes by a lentiviral system. The cells were plated in 24-well plates and transfected with 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Luciferase activities were measured. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. ns, nonsignificant. (B) 293T-EGFP cells were generated, in which the CMV promoter driving EGFP was randomly integrated into cellular chromosomes by a lentiviral system. The cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR. GFP and β-actin protein were detected by Western blot analyses. (C) 293T cells were plated in 12-well plates and cotransfected with 0.5 μg pFlag-IRAK1 and 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Flag-IRAK1 and GAPDH protein levels were determined by Western blot analyses. (D) 293T cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Endogenous IRAK1 and GAPDH were detected by Western blot analyses. (E) HepG2 cells were plated in 12-well plates and cotransfected with 0.5 μg pCAGGS-HA-IFNAR1 and 0.5 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. HA-IFNAR1 and GAPDH were detected by Western blot analyses. (F) HepG2 cells were plated in 12-well plates and transfected with 1 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. Endogenous IFNAR1 and GAPDH were detected by Western blot analyses. (G) RD cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 48 h, and infected with EV71 at an MOI of 5 for 6 h. EV71 VP1, HA-PJA1B, and GAPDH were detected by Western blot analyses. (H) 293T cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 24 h, and infected with VSV-GFP at an MOI of 1 for 12 h. GFP, PJA1B, and GAPDH were detected by Western blot analyses.
    Figure Legend Snippet: PJA1 has no effect on chromosome-integrated genes and RNA viruses. (A) 293T-Luc cells were generated, in which the CMV promoter driving Luc was randomly integrated into cell chromosomes by a lentiviral system. The cells were plated in 24-well plates and transfected with 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Luciferase activities were measured. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. ns, nonsignificant. (B) 293T-EGFP cells were generated, in which the CMV promoter driving EGFP was randomly integrated into cellular chromosomes by a lentiviral system. The cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR. GFP and β-actin protein were detected by Western blot analyses. (C) 293T cells were plated in 12-well plates and cotransfected with 0.5 μg pFlag-IRAK1 and 0.5 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Flag-IRAK1 and GAPDH protein levels were determined by Western blot analyses. (D) 293T cells were plated in 12-well plates and transfected with 1 μg pCAGGS, pCAGGS-HA-PJA1, or pCAGGS-HA-PJA1ΔR for 48 h. Endogenous IRAK1 and GAPDH were detected by Western blot analyses. (E) HepG2 cells were plated in 12-well plates and cotransfected with 0.5 μg pCAGGS-HA-IFNAR1 and 0.5 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. HA-IFNAR1 and GAPDH were detected by Western blot analyses. (F) HepG2 cells were plated in 12-well plates and transfected with 1 μg pcDNA3.1, pcDNA3.1-PJA1, or pcDNA3.1-PJA1ΔR for 48 h. Endogenous IFNAR1 and GAPDH were detected by Western blot analyses. (G) RD cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 48 h, and infected with EV71 at an MOI of 5 for 6 h. EV71 VP1, HA-PJA1B, and GAPDH were detected by Western blot analyses. (H) 293T cells were plated in 6-well plates, transfected with pCAGGS-HA-PJA1B at different concentrations (0, 0.5, 1, and 2 μg) for 24 h, and infected with VSV-GFP at an MOI of 1 for 12 h. GFP, PJA1B, and GAPDH were detected by Western blot analyses.

    Techniques Used: Generated, Transfection, Luciferase, Western Blot, Infection

    17) Product Images from "The m6A eraser FTO facilitates proliferation and migration of human cervical cancer cells"

    Article Title: The m6A eraser FTO facilitates proliferation and migration of human cervical cancer cells

    Journal: Cancer Cell International

    doi: 10.1186/s12935-019-1045-1

    The m 6 A demethylase activity is required for FTO to play its oncogenic function. a Enforced FTO or FTO-mut expression in Hela cells. FTO-mut carries two point- mutations, H231A and D233A, which disrupt the enzymatic activity of FTO. GAPDH was used as a loading control; b m 6 A dot blot assays of Hela cells with enforced FTO or FTO-mut expression. MB, methylene blue staining (as loading control); c effects of FTO or mutant FTO overexpression on Hela cells’ growth/proliferation, *** P
    Figure Legend Snippet: The m 6 A demethylase activity is required for FTO to play its oncogenic function. a Enforced FTO or FTO-mut expression in Hela cells. FTO-mut carries two point- mutations, H231A and D233A, which disrupt the enzymatic activity of FTO. GAPDH was used as a loading control; b m 6 A dot blot assays of Hela cells with enforced FTO or FTO-mut expression. MB, methylene blue staining (as loading control); c effects of FTO or mutant FTO overexpression on Hela cells’ growth/proliferation, *** P

    Techniques Used: Activity Assay, Expressing, Dot Blot, Staining, Mutagenesis, Over Expression

    18) Product Images from "Nearly complete deletion of BubR1 causes microcephaly through shortened mitosis and massive cell death"

    Article Title: Nearly complete deletion of BubR1 causes microcephaly through shortened mitosis and massive cell death

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddz022

    Depletion of BubR1 during neurogenesis is nearly complete in BubR1 CKO. ( A – C ) At E12.5, minimal BubR1 is present at the ventricular surface in BubR1 H/H neuroepithelium ( B , inset, arrows), where it is located on the chromosomes of PH3+ cells undergoing mitosis (arrows). It is almost completely absent in BubR1 CKO (C) but highly enriched in WT (A) . Arrowheads indicate nonspecific or auto-fluorescence signals from blood cells (B and C). ( D ) Western blot of E15.5 cortices confirms significant reduction of BubR1 expression in BubR1 H/H and BubR1 CKO compared to WT. ( E ) The graph compares the relative levels of BubR1 protein in the BubR1 H/H and BubR1 CKO to the WT level after normalization with GAPDH. ** P
    Figure Legend Snippet: Depletion of BubR1 during neurogenesis is nearly complete in BubR1 CKO. ( A – C ) At E12.5, minimal BubR1 is present at the ventricular surface in BubR1 H/H neuroepithelium ( B , inset, arrows), where it is located on the chromosomes of PH3+ cells undergoing mitosis (arrows). It is almost completely absent in BubR1 CKO (C) but highly enriched in WT (A) . Arrowheads indicate nonspecific or auto-fluorescence signals from blood cells (B and C). ( D ) Western blot of E15.5 cortices confirms significant reduction of BubR1 expression in BubR1 H/H and BubR1 CKO compared to WT. ( E ) The graph compares the relative levels of BubR1 protein in the BubR1 H/H and BubR1 CKO to the WT level after normalization with GAPDH. ** P

    Techniques Used: Fluorescence, Western Blot, Expressing

    19) Product Images from "Role of the Wnt/β-catenin signaling pathway in inducing apoptosis and renal fibrosis in 5/6-nephrectomized rats"

    Article Title: Role of the Wnt/β-catenin signaling pathway in inducing apoptosis and renal fibrosis in 5/6-nephrectomized rats

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.6461

    Apoptosis is increased in kidneys from CKD rats. (A) Representative images of TUNEL staining (red) in kidney sections from control (sham-operated) and CKD (5/6-nephrectomized) rats. Nuclei were counterstained with DAPI (blue). Original magnification, ×400. (B) Representative western blot images and (C) quantification of cleaved caspase-3, Bax and Bcl-2 protein expression levels in kidney tissues from control and CKD groups. GAPDH served as a loading control. Data are expressed as the mean ± standard error (n=8 per group). *P
    Figure Legend Snippet: Apoptosis is increased in kidneys from CKD rats. (A) Representative images of TUNEL staining (red) in kidney sections from control (sham-operated) and CKD (5/6-nephrectomized) rats. Nuclei were counterstained with DAPI (blue). Original magnification, ×400. (B) Representative western blot images and (C) quantification of cleaved caspase-3, Bax and Bcl-2 protein expression levels in kidney tissues from control and CKD groups. GAPDH served as a loading control. Data are expressed as the mean ± standard error (n=8 per group). *P

    Techniques Used: TUNEL Assay, Staining, Western Blot, Expressing

    20) Product Images from "Tristetraprolin Overexpression in Gastric Cancer Cells Suppresses PD-L1 Expression and Inhibits Tumor Progression by Enhancing Antitumor Immunity"

    Article Title: Tristetraprolin Overexpression in Gastric Cancer Cells Suppresses PD-L1 Expression and Inhibits Tumor Progression by Enhancing Antitumor Immunity

    Journal: Molecules and Cells

    doi: 10.14348/molcells.2018.0040

    TTP overexpression reduced cell survival and promoted apoptosis in both MGC-803 and BGC-823 cells. MGC-803 and BGC-823 cells were transfected with pcDNA-TTP or empty vector pcDNA3.1 (+) (A) Relative expression of TTP mRNA in MGC-803/TTP and BGC-823/TTP cell lines and corresponding control group was examined by qRT-PCR. An empty vector ctr clone was used as the control. (B) The viability rate of GC cells was measured by trypan blue dye exclusion assay. (C) Expression of TTP protein level was examined by western blotting. Bcl-2 and cleavage of caspase 3 expression in MGC-803/TTP and BGC-823/TTP and the corresponding control group were analyzed by western blotting. GAPDH and β-actin were used as internal controls for qRT-PCR and western blotting analysis, respectively. (D) Quantifications of western blotting results was processed by Image J software. All data were represented as the mean ± SD of three independent experiments. *P
    Figure Legend Snippet: TTP overexpression reduced cell survival and promoted apoptosis in both MGC-803 and BGC-823 cells. MGC-803 and BGC-823 cells were transfected with pcDNA-TTP or empty vector pcDNA3.1 (+) (A) Relative expression of TTP mRNA in MGC-803/TTP and BGC-823/TTP cell lines and corresponding control group was examined by qRT-PCR. An empty vector ctr clone was used as the control. (B) The viability rate of GC cells was measured by trypan blue dye exclusion assay. (C) Expression of TTP protein level was examined by western blotting. Bcl-2 and cleavage of caspase 3 expression in MGC-803/TTP and BGC-823/TTP and the corresponding control group were analyzed by western blotting. GAPDH and β-actin were used as internal controls for qRT-PCR and western blotting analysis, respectively. (D) Quantifications of western blotting results was processed by Image J software. All data were represented as the mean ± SD of three independent experiments. *P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR, Exclusion Assay, Western Blot, Software

    21) Product Images from "Molecular mechanisms in vascular injury induced by hypertension: Expression and role of microRNA-34a"

    Article Title: Molecular mechanisms in vascular injury induced by hypertension: Expression and role of microRNA-34a

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.5216

    Tigf2 is a target of miR-34a. (A) The results of bioinformatics. Wild-type or mutant Tigf2 3′UTR luciferase reporter constructs were cotransfected with miR-34a mimic or NC scramble-miR, and luciferase activities were assayed 24 h post-transfection. (B) Human umbilical vein endothelial cells were transfected with an miR-34a inhibitor or NC scramble-miR. Cell lysates were prepared and assessed by western blot analysis to detect the expression of TIGF2 and GAPDH (internal control).*P
    Figure Legend Snippet: Tigf2 is a target of miR-34a. (A) The results of bioinformatics. Wild-type or mutant Tigf2 3′UTR luciferase reporter constructs were cotransfected with miR-34a mimic or NC scramble-miR, and luciferase activities were assayed 24 h post-transfection. (B) Human umbilical vein endothelial cells were transfected with an miR-34a inhibitor or NC scramble-miR. Cell lysates were prepared and assessed by western blot analysis to detect the expression of TIGF2 and GAPDH (internal control).*P

    Techniques Used: Mutagenesis, Luciferase, Construct, Transfection, Western Blot, Expressing

    22) Product Images from "Anti-tumoral potential of MDA19 in human osteosarcoma via suppressing PI3K/Akt/mTOR signaling pathway"

    Article Title: Anti-tumoral potential of MDA19 in human osteosarcoma via suppressing PI3K/Akt/mTOR signaling pathway

    Journal: Bioscience Reports

    doi: 10.1042/BSR20181501

    The effect of apoptosis in OS cells administrated with MDA19 was tested by flow cytometry after Annexin V-FITC/PI staining and Western blot assay ( A ) The change of apoptotic rate in U2OS and MG-63 cells after incubation of MDA19. ( B ) Western blot analysis was performed to test a series of antibodies (Bcl-2, Bax, Cleaved Caspase-3, and Gapdh). All values are expressed as mean ± S.D. ** P
    Figure Legend Snippet: The effect of apoptosis in OS cells administrated with MDA19 was tested by flow cytometry after Annexin V-FITC/PI staining and Western blot assay ( A ) The change of apoptotic rate in U2OS and MG-63 cells after incubation of MDA19. ( B ) Western blot analysis was performed to test a series of antibodies (Bcl-2, Bax, Cleaved Caspase-3, and Gapdh). All values are expressed as mean ± S.D. ** P

    Techniques Used: Flow Cytometry, Cytometry, Staining, Western Blot, Incubation

    23) Product Images from "Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis"

    Article Title: Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0729-0

    MiR-23b-3p could enhance matrix degradation by means of regulating activity of p38 MAPK in human chondrocytes under TNF-α treatment. a , b SW1353 cells were transfected by mimic miR-23b-3p ( a ) or anti-miR-23b-3p sequence ( b ) with or without TNF-α for 24 h, and the protein expression of phosphorylation form of p38 MAPK (p-p38), total p38 MAPK, and MMP13 was detected by western blotting. Asterisk (*): compared with the mimic NC ( a ) or anti-NC group ( b ). c SW1353 cells were treated with mimic miR-23b-3p with or without p38 MAPK inhibitor SB203580 (10 μM) under stimulation of TNF-α. The protein expression of MMP13 was determined by western blotting. Asterisk (*): compared with the mimic NC group. d Interfering efficiency of siRNAs against p38 MAPK was determined by western blotting under 50 nM p38 siRNA, 50 nM negative control (NC), and Mock (transfection regent only) transfection for 48 h. e Under stimulation with TNF-α, SW1353 cells were transfected by mimic miR-23b-3p under treatment with si- p38 mixture (containing three siRNA target sequences), and p-p38, total p38, and MMP13 were determined by western blotting. Asterisk (*): compared with mimic NC or si-NC. Each relative expression of phosphorylation form was normalized by the total form. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
    Figure Legend Snippet: MiR-23b-3p could enhance matrix degradation by means of regulating activity of p38 MAPK in human chondrocytes under TNF-α treatment. a , b SW1353 cells were transfected by mimic miR-23b-3p ( a ) or anti-miR-23b-3p sequence ( b ) with or without TNF-α for 24 h, and the protein expression of phosphorylation form of p38 MAPK (p-p38), total p38 MAPK, and MMP13 was detected by western blotting. Asterisk (*): compared with the mimic NC ( a ) or anti-NC group ( b ). c SW1353 cells were treated with mimic miR-23b-3p with or without p38 MAPK inhibitor SB203580 (10 μM) under stimulation of TNF-α. The protein expression of MMP13 was determined by western blotting. Asterisk (*): compared with the mimic NC group. d Interfering efficiency of siRNAs against p38 MAPK was determined by western blotting under 50 nM p38 siRNA, 50 nM negative control (NC), and Mock (transfection regent only) transfection for 48 h. e Under stimulation with TNF-α, SW1353 cells were transfected by mimic miR-23b-3p under treatment with si- p38 mixture (containing three siRNA target sequences), and p-p38, total p38, and MMP13 were determined by western blotting. Asterisk (*): compared with mimic NC or si-NC. Each relative expression of phosphorylation form was normalized by the total form. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value

    Techniques Used: Activity Assay, Transfection, Sequencing, Expressing, Western Blot, Negative Control, MANN-WHITNEY

    HS6ST2 could regulate the matrix degradation depending on the activity of p38 MAPK. a SW1353 cells were transfected by si -HS6ST2 or negative control (si-NC) with or without TNF-α for 24 h, and the protein expression of p-p38 and p38 was detected by western blotting. Asterisk (*): compared with the si-NC group. b Under stimulation with TNF-α, SW1353 cells were transfected by empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) under treatment with mimic miR-23b-3p, and p-p38 and total p38 were determined by western blotting. Asterisk (*): compared with the mimic NC group. c SW1353 cells were transfected with HS6ST2 siRNA with or without p38 MAPK inhibitor SB203580 (10 μM) after treatment of TNF-α, and the protein expression of MMP13 was determined by western blotting. Each relative expression of phosphorylation form was normalized by the total form. d Schematic representation of miR-23b-3p–HS6ST2 axis-mediated catabolic effects in human chondrocyte. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
    Figure Legend Snippet: HS6ST2 could regulate the matrix degradation depending on the activity of p38 MAPK. a SW1353 cells were transfected by si -HS6ST2 or negative control (si-NC) with or without TNF-α for 24 h, and the protein expression of p-p38 and p38 was detected by western blotting. Asterisk (*): compared with the si-NC group. b Under stimulation with TNF-α, SW1353 cells were transfected by empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) under treatment with mimic miR-23b-3p, and p-p38 and total p38 were determined by western blotting. Asterisk (*): compared with the mimic NC group. c SW1353 cells were transfected with HS6ST2 siRNA with or without p38 MAPK inhibitor SB203580 (10 μM) after treatment of TNF-α, and the protein expression of MMP13 was determined by western blotting. Each relative expression of phosphorylation form was normalized by the total form. d Schematic representation of miR-23b-3p–HS6ST2 axis-mediated catabolic effects in human chondrocyte. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value

    Techniques Used: Activity Assay, Transfection, Negative Control, Expressing, Western Blot, Plasmid Preparation, MANN-WHITNEY

    MiR-23b-3p could enhance matrix degradation in human chondrocytes via regulating HS6ST2. a , b SW1353 cells transfected with 50 nM HS6ST2 siRNA mixture (containing three target sequences, si- HS6ST2 ) or negative control (si-NC) were stimulated by 10 ng/ml TNF-α for 24 h. Protein level of HS6ST2 and MMP13 were assayed by western blotting ( a ) and matrix content of chondrocytes was determined by toluidine blue staining ( b ). Asterisk (*): compared with the si-NC group. c , d SW1353 cells transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for 24 h were stimulated by TNF-α for another 24 h. The protein expression of MMP13 was assayed by western blotting ( c ) and matrix content of chondrocytes was determined by toluidine blue staining ( d ). Asterisk (*): compared with the FLAG-Ctrl group. e , f Under stimulation with TNF-α, SW1353 cells were treated with mimic NC or mimic miR-23b-3p for 24 h and then transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for another 24 h to observe the rescuing effect of mimic miR-23b-3p in MMP13 expression ( e ) and matrix content ( f ). Asterisk (*): compared with the mimic NC group. GAPDH was used as internal controls in western blotting detection. In toluidine blue staining results, scale bar, 100 μm. Lower panel, statistical analysis of average optical density of matrix staining of toluidine blue. Bars represent standard error of the mean (SEM) from three independent experiments. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
    Figure Legend Snippet: MiR-23b-3p could enhance matrix degradation in human chondrocytes via regulating HS6ST2. a , b SW1353 cells transfected with 50 nM HS6ST2 siRNA mixture (containing three target sequences, si- HS6ST2 ) or negative control (si-NC) were stimulated by 10 ng/ml TNF-α for 24 h. Protein level of HS6ST2 and MMP13 were assayed by western blotting ( a ) and matrix content of chondrocytes was determined by toluidine blue staining ( b ). Asterisk (*): compared with the si-NC group. c , d SW1353 cells transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for 24 h were stimulated by TNF-α for another 24 h. The protein expression of MMP13 was assayed by western blotting ( c ) and matrix content of chondrocytes was determined by toluidine blue staining ( d ). Asterisk (*): compared with the FLAG-Ctrl group. e , f Under stimulation with TNF-α, SW1353 cells were treated with mimic NC or mimic miR-23b-3p for 24 h and then transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for another 24 h to observe the rescuing effect of mimic miR-23b-3p in MMP13 expression ( e ) and matrix content ( f ). Asterisk (*): compared with the mimic NC group. GAPDH was used as internal controls in western blotting detection. In toluidine blue staining results, scale bar, 100 μm. Lower panel, statistical analysis of average optical density of matrix staining of toluidine blue. Bars represent standard error of the mean (SEM) from three independent experiments. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value

    Techniques Used: Transfection, Negative Control, Western Blot, Staining, Plasmid Preparation, Expressing, MANN-WHITNEY

    24) Product Images from "Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress"

    Article Title: Acetylation of oxidized base repair-initiating NEIL1 DNA glycosylase required for chromatin-bound repair complex formation in the human genome increases cellular resistance to oxidative stress

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2018.04.001

    Characterization of α-AcNEIL1 and detection of AcNEIL1 in cells (A) 25-200 ng of unmodified (−Ac) or in vitro acetylated (+Ac) recombinant (Rec) WT NEIL1 was subjected to SDS PAGE for Western analysis with α-AcNEIL1 and α-NEIL1. (B) Western analysis of cell extracts from control vs. siRNA-mediated NEIL1 downregulated HT29 cells for AcNEIL1, total NEIL1 and GAPDH levels. (C and D) Western analysis of (C) soluble nuclear extracts and chromatin extracts of HEK293, and (D) chromatin extracts of HCT116 cells after GOx treatment (25 ng/ml) for the indicated times for AcNEIL1, total NEIL1 and H3 levels; quantitation of AcNEIL1 level (ImageJ software) shown in the associated histograms.
    Figure Legend Snippet: Characterization of α-AcNEIL1 and detection of AcNEIL1 in cells (A) 25-200 ng of unmodified (−Ac) or in vitro acetylated (+Ac) recombinant (Rec) WT NEIL1 was subjected to SDS PAGE for Western analysis with α-AcNEIL1 and α-NEIL1. (B) Western analysis of cell extracts from control vs. siRNA-mediated NEIL1 downregulated HT29 cells for AcNEIL1, total NEIL1 and GAPDH levels. (C and D) Western analysis of (C) soluble nuclear extracts and chromatin extracts of HEK293, and (D) chromatin extracts of HCT116 cells after GOx treatment (25 ng/ml) for the indicated times for AcNEIL1, total NEIL1 and H3 levels; quantitation of AcNEIL1 level (ImageJ software) shown in the associated histograms.

    Techniques Used: In Vitro, Recombinant, SDS Page, Western Blot, Quantitation Assay, Software

    25) Product Images from "Lactobacillus rhamnosus GG Affects Microbiota and Suppresses Autophagy in the Intestines of Pigs Challenged with Salmonella Infantis"

    Article Title: Lactobacillus rhamnosus GG Affects Microbiota and Suppresses Autophagy in the Intestines of Pigs Challenged with Salmonella Infantis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02705

    LGG pretreatment attenuated S . Infantis-induced autophagy and increased EGFR and Akt activation in the ileum of pigs. Representative panels of LC3A/B-I and LC3A/B-II (A) , p-EGFR and total-EGFR (B) , and p-Akt and total-Akt (C) proteins in the jejunum and ileum tissues collected from pigs at 10 days after S . Infantis challenge (left panel). Each band represents an individual pig. The results are presented as the ratios of the band intensities of LC3A/B-II to GAPDH, p-Akt to total-Akt and p-EGFR to total-EGFR (right panel). (D) Immunofluorescence analysis of the recovered S . Infantis (mCherry) in the liver. Frozen sections of liver from pigs challenged with S . Infantis were stained with anti-pig α-tubulin (green) and DAPI (blue). Scare bars, 50 μm. (E) The number of live S . Infantis recovered from the livers of the pigs was determined. The data are presented as mean ± SEM ( n = 7 per group). * P
    Figure Legend Snippet: LGG pretreatment attenuated S . Infantis-induced autophagy and increased EGFR and Akt activation in the ileum of pigs. Representative panels of LC3A/B-I and LC3A/B-II (A) , p-EGFR and total-EGFR (B) , and p-Akt and total-Akt (C) proteins in the jejunum and ileum tissues collected from pigs at 10 days after S . Infantis challenge (left panel). Each band represents an individual pig. The results are presented as the ratios of the band intensities of LC3A/B-II to GAPDH, p-Akt to total-Akt and p-EGFR to total-EGFR (right panel). (D) Immunofluorescence analysis of the recovered S . Infantis (mCherry) in the liver. Frozen sections of liver from pigs challenged with S . Infantis were stained with anti-pig α-tubulin (green) and DAPI (blue). Scare bars, 50 μm. (E) The number of live S . Infantis recovered from the livers of the pigs was determined. The data are presented as mean ± SEM ( n = 7 per group). * P

    Techniques Used: Activation Assay, Immunofluorescence, Staining

    LGG pretreatment attenuated S . Infantis-induced autophagy and promoted EGFR and Akt activation in the IPEC-J2 cell monolayers. (A ) Autophagy in IPEC-J2 cells infected with S . Infantis was observed using transmission electron microscopy. Black arrows indicate S . Infantis and black arrowheads indicate single or double membrane of autophagosomes. Representative panels of LC3A/B-I and LC3A/B-II (B) , p-EGFR and total-EGFR (C) , and p-Akt and total-Akt (D) proteins in IPEC-J2 cells collected at the indicated time points after S . Infantis challenge (left panel). Each band represents an individual pig. The results are presented as the ratios of the band intensities of LC3A/B-II to GAPDH, p-Akt to total-Akt and p-EGFR to total-EGFR (right panel). The data are presented as mean ± SEM of three independent experiments. * P
    Figure Legend Snippet: LGG pretreatment attenuated S . Infantis-induced autophagy and promoted EGFR and Akt activation in the IPEC-J2 cell monolayers. (A ) Autophagy in IPEC-J2 cells infected with S . Infantis was observed using transmission electron microscopy. Black arrows indicate S . Infantis and black arrowheads indicate single or double membrane of autophagosomes. Representative panels of LC3A/B-I and LC3A/B-II (B) , p-EGFR and total-EGFR (C) , and p-Akt and total-Akt (D) proteins in IPEC-J2 cells collected at the indicated time points after S . Infantis challenge (left panel). Each band represents an individual pig. The results are presented as the ratios of the band intensities of LC3A/B-II to GAPDH, p-Akt to total-Akt and p-EGFR to total-EGFR (right panel). The data are presented as mean ± SEM of three independent experiments. * P

    Techniques Used: Activation Assay, Infection, Transmission Assay, Electron Microscopy

    26) Product Images from "Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis"

    Article Title: Proteasome inhibitor bortezomib is a novel therapeutic agent for focal radiation-induced osteoporosis

    Journal: The FASEB Journal

    doi: 10.1096/fj.201700375R

    Proteasome inhibition upregulates NHEJ pathway proteins to stimulate DNA repair. A ) MG132 (20 nM) increases Ku70 and DNA-PKc amounts in UMR106 cells after radiation, as shown by immunoblot analysis. The band intensities listed below the blot were quantified by densitometric analysis and normalized against GAPDH. B ) Immunofluorescence staining of Ku70 (green) and DNA-PKc (white) in irradiated mouse calvarial organ culture at 6 h in the presence or absence of MG132. Merged images are shown with white arrows indicating cells having both proteins and a yellow arrow indicating a cell having only Ku70 ( n = 3 calvariae/group). C ) The percentage of Ku70 + or DNA-PKc + osteoblasts was quantified. $ P
    Figure Legend Snippet: Proteasome inhibition upregulates NHEJ pathway proteins to stimulate DNA repair. A ) MG132 (20 nM) increases Ku70 and DNA-PKc amounts in UMR106 cells after radiation, as shown by immunoblot analysis. The band intensities listed below the blot were quantified by densitometric analysis and normalized against GAPDH. B ) Immunofluorescence staining of Ku70 (green) and DNA-PKc (white) in irradiated mouse calvarial organ culture at 6 h in the presence or absence of MG132. Merged images are shown with white arrows indicating cells having both proteins and a yellow arrow indicating a cell having only Ku70 ( n = 3 calvariae/group). C ) The percentage of Ku70 + or DNA-PKc + osteoblasts was quantified. $ P

    Techniques Used: Inhibition, Non-Homologous End Joining, Immunofluorescence, Staining, Irradiation, Organ Culture

    27) Product Images from "PTEN inhibitor VO-OHpic attenuates GC-associated endothelial progenitor cell dysfunction and osteonecrosis of the femoral head via activating Nrf2 signaling and inhibiting mitochondrial apoptosis pathway"

    Article Title: PTEN inhibitor VO-OHpic attenuates GC-associated endothelial progenitor cell dysfunction and osteonecrosis of the femoral head via activating Nrf2 signaling and inhibiting mitochondrial apoptosis pathway

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-020-01658-y

    Nrf2 activation is required for VO-OHpic-mediated anti-MPS effects in EPCs. a Relative mRNA expressions of Nrf2 as determined by RT-qPCR 2 days after siNrf2 transfection. b EPCs transfected with or without siNrf2 for 24 h were then treated with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic for 48 h. Nrf2, NQO-1, HO-1, and Trx protein levels were determined by western blot analysis. c Band density ratios of Nrf2, NQO-1, HO-1, and Trx to GAPDH in the western blots were quantified by densitometry. d The cleaved caspase 3 protein level was also determined by western blot analysis. e Band density ratios of cleaved caspase 3 to GAPDH in the western blots were quantified by densitometry. f Flow cytometric analysis of EPCs transfected with or without siNrf2 were stained with Annexin V-FITC/PI after treatment with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic for 48 h. g Percentage of apoptosis rates was expressed as means ± SD. h The VEGF protein level was determined by western blot analysis. i Band density ratios of VEGF to GAPDH in the western blots were quantified by densitometry. j Representative images of tube formation of EPCs in 12 h. EPCs were pre-transfected with and without siNrf2 for 24 h and then treated with 50-μM MPS combined with 1-μM VO-OHpic before seeded into Matrigel. k Representative images of EPCs transfected with or without siNrf2 with intracellular ROS stained by the fluorescence probe DCFH-DA after treatment for 48 h. l Flow cytometric analysis of ROS production after staining with DCFH-DA. m Bar graphs showing the mean fluorescence intensity (MFI) of ROS levels in EPCs. Data are shown as means ± SD. All experiments were repeated for three times; ∗ P
    Figure Legend Snippet: Nrf2 activation is required for VO-OHpic-mediated anti-MPS effects in EPCs. a Relative mRNA expressions of Nrf2 as determined by RT-qPCR 2 days after siNrf2 transfection. b EPCs transfected with or without siNrf2 for 24 h were then treated with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic for 48 h. Nrf2, NQO-1, HO-1, and Trx protein levels were determined by western blot analysis. c Band density ratios of Nrf2, NQO-1, HO-1, and Trx to GAPDH in the western blots were quantified by densitometry. d The cleaved caspase 3 protein level was also determined by western blot analysis. e Band density ratios of cleaved caspase 3 to GAPDH in the western blots were quantified by densitometry. f Flow cytometric analysis of EPCs transfected with or without siNrf2 were stained with Annexin V-FITC/PI after treatment with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic for 48 h. g Percentage of apoptosis rates was expressed as means ± SD. h The VEGF protein level was determined by western blot analysis. i Band density ratios of VEGF to GAPDH in the western blots were quantified by densitometry. j Representative images of tube formation of EPCs in 12 h. EPCs were pre-transfected with and without siNrf2 for 24 h and then treated with 50-μM MPS combined with 1-μM VO-OHpic before seeded into Matrigel. k Representative images of EPCs transfected with or without siNrf2 with intracellular ROS stained by the fluorescence probe DCFH-DA after treatment for 48 h. l Flow cytometric analysis of ROS production after staining with DCFH-DA. m Bar graphs showing the mean fluorescence intensity (MFI) of ROS levels in EPCs. Data are shown as means ± SD. All experiments were repeated for three times; ∗ P

    Techniques Used: Activation Assay, Quantitative RT-PCR, Transfection, Western Blot, Staining, Fluorescence

    VO-OHpic exerts protective effects on MPS-regulated angiogenesis of EPCs. a Representative images of tube formation of EPCs after treatment with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic for 48 h. b VEGF protein concentration in the culture medium of EPCs as determined by ELISA after treatment for 48 h. c Wound healing assay was performed and representative images were taken after 12 h and 24 h in EPCs treated with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic. d The wound closure rate was measured and relatively compared to the control group with no treatment of MPS and VO-OHpic. e VEGF, VEGFR1, and VEGFR2 protein levels were determined by western blot analysis after treatment for 48 h. f Band density ratios of VEGF, VEGFR1, and VEGFR2 to GAPDH in the western blot analysis were quantified by densitometry. All experiments were repeated for three times; ∗ P
    Figure Legend Snippet: VO-OHpic exerts protective effects on MPS-regulated angiogenesis of EPCs. a Representative images of tube formation of EPCs after treatment with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic for 48 h. b VEGF protein concentration in the culture medium of EPCs as determined by ELISA after treatment for 48 h. c Wound healing assay was performed and representative images were taken after 12 h and 24 h in EPCs treated with 50-μM MPS or 50-μM MPS combined with 1-μM VO-OHpic. d The wound closure rate was measured and relatively compared to the control group with no treatment of MPS and VO-OHpic. e VEGF, VEGFR1, and VEGFR2 protein levels were determined by western blot analysis after treatment for 48 h. f Band density ratios of VEGF, VEGFR1, and VEGFR2 to GAPDH in the western blot analysis were quantified by densitometry. All experiments were repeated for three times; ∗ P

    Techniques Used: Protein Concentration, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Western Blot

    28) Product Images from "Analysis of Differentially Expressed Genes in a Chinese Cohort of Esophageal Squamous Cell Carcinoma"

    Article Title: Analysis of Differentially Expressed Genes in a Chinese Cohort of Esophageal Squamous Cell Carcinoma

    Journal: Journal of Cancer

    doi: 10.7150/jca.40850

    Real-time PCR and Western blotting analyses of CLIC2, CLIC3 and CLIC4 in ESCCs and paired NTs. (A-C): CLIC2 (A), CLIC3 (B) and CLIC4 (C) mRNA assays by qPCR between ESCCs and paired NTs. (D-F): Representative CLIC2 (D), CLIC3 (E) and CLIC4 (F) protein assays by Western blotting in ESCCs and paired NTs, GAPDH served as loading control. ( G-I): Statistical analyses of CLIC2 (G), CLIC3 (H) and CLIC4 (I) protein levels between ESCCs and paired NTs.
    Figure Legend Snippet: Real-time PCR and Western blotting analyses of CLIC2, CLIC3 and CLIC4 in ESCCs and paired NTs. (A-C): CLIC2 (A), CLIC3 (B) and CLIC4 (C) mRNA assays by qPCR between ESCCs and paired NTs. (D-F): Representative CLIC2 (D), CLIC3 (E) and CLIC4 (F) protein assays by Western blotting in ESCCs and paired NTs, GAPDH served as loading control. ( G-I): Statistical analyses of CLIC2 (G), CLIC3 (H) and CLIC4 (I) protein levels between ESCCs and paired NTs.

    Techniques Used: Real-time Polymerase Chain Reaction, Western Blot

    29) Product Images from "PTEN-knockdown disrupts the morphology, growth pattern and function of Nthy-Ori 3-1 cells by downregulating PAX8 expression"

    Article Title: PTEN-knockdown disrupts the morphology, growth pattern and function of Nthy-Ori 3-1 cells by downregulating PAX8 expression

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.11028

    PTEN affects Nthy-Ori 3-1 cell function by regulating PAX8. (A) Expression of the thyroid-specific proteins TG, TPO and NIS in shControl, shPTEN and shPTEN+PAX8 groups of cells detected using western blot analysis. GAPDH served as an internal control. (B) Iodide uptake ability was measured at different times following the addition of 125 I. Data are presented as the mean ± standard error of mean from at least three different experiments. *P
    Figure Legend Snippet: PTEN affects Nthy-Ori 3-1 cell function by regulating PAX8. (A) Expression of the thyroid-specific proteins TG, TPO and NIS in shControl, shPTEN and shPTEN+PAX8 groups of cells detected using western blot analysis. GAPDH served as an internal control. (B) Iodide uptake ability was measured at different times following the addition of 125 I. Data are presented as the mean ± standard error of mean from at least three different experiments. *P

    Techniques Used: Cell Function Assay, Expressing, Western Blot

    30) Product Images from "Leucine-rich repeat containing 4 act as an autophagy inhibitor that restores sensitivity of glioblastoma to temozolomide"

    Article Title: Leucine-rich repeat containing 4 act as an autophagy inhibitor that restores sensitivity of glioblastoma to temozolomide

    Journal: Oncogene

    doi: 10.1038/s41388-020-1312-6

    LRRC4 promoted the degradation of DEPTOR protein, and re-expression of DEPTOR restored autophagy activation. a Representative western blotting analysis of LRRC4, DEPTOR, LC3B and GAPDH in U251, PG1 and PG2 cells with or without transfection with LRRC4. Numbers represent the relative ratio of DEPTOR/LC3B and GAPDH. b Western blotting analysis was used to measure of the half-life of DEPTOR after treatment with cycloheximide in U251 cells with (down) or without (upper) LRRC4 transfection. Cells were lysed and protein was extracted at 0, 4, 8, 16, 24 h after cycloheximide treatment. c DEPTOR ubiquitination was assessed by an anti-GFP antibody in the presence of MG132 when GFP-DEPTOR, HA-ubiquitin, and LRRC4 or vector were co-transfected into U251 cells. d Representative western blotting analysis of LRRC4, p-MTOR, MTOR, p-ULK1, ULK1, p-S6K, S6K and Tubulin in U251 and PG2 cells with or without transfection with LRRC4. e Representative western blotting analysis of LRRC4, p-MTOR, p-S6K, S6K, P62, LC3B and GAPDH in U251 cells, U251 cells with or without LRRC4 transfection were treated with dimethylsulfoxide (DMSO) or Rapamycin. f Representative western blotting analysis of LRRC4, DEPTOR, LC3B and GAPDH in U251 cells, U251 cells with stable expression LRRC4 and LRRC4-expressing U251 cells with ectopic expression of DEPTOR. g Autophagosomes were observed by transmission electron microscopy in U251 cells, U251 cells with stable expression LRRC4 and LRRC4-expressing U251 cells with ectopic expression of DEPTOR.
    Figure Legend Snippet: LRRC4 promoted the degradation of DEPTOR protein, and re-expression of DEPTOR restored autophagy activation. a Representative western blotting analysis of LRRC4, DEPTOR, LC3B and GAPDH in U251, PG1 and PG2 cells with or without transfection with LRRC4. Numbers represent the relative ratio of DEPTOR/LC3B and GAPDH. b Western blotting analysis was used to measure of the half-life of DEPTOR after treatment with cycloheximide in U251 cells with (down) or without (upper) LRRC4 transfection. Cells were lysed and protein was extracted at 0, 4, 8, 16, 24 h after cycloheximide treatment. c DEPTOR ubiquitination was assessed by an anti-GFP antibody in the presence of MG132 when GFP-DEPTOR, HA-ubiquitin, and LRRC4 or vector were co-transfected into U251 cells. d Representative western blotting analysis of LRRC4, p-MTOR, MTOR, p-ULK1, ULK1, p-S6K, S6K and Tubulin in U251 and PG2 cells with or without transfection with LRRC4. e Representative western blotting analysis of LRRC4, p-MTOR, p-S6K, S6K, P62, LC3B and GAPDH in U251 cells, U251 cells with or without LRRC4 transfection were treated with dimethylsulfoxide (DMSO) or Rapamycin. f Representative western blotting analysis of LRRC4, DEPTOR, LC3B and GAPDH in U251 cells, U251 cells with stable expression LRRC4 and LRRC4-expressing U251 cells with ectopic expression of DEPTOR. g Autophagosomes were observed by transmission electron microscopy in U251 cells, U251 cells with stable expression LRRC4 and LRRC4-expressing U251 cells with ectopic expression of DEPTOR.

    Techniques Used: Expressing, Activation Assay, Western Blot, Transfection, Plasmid Preparation, Transmission Assay, Electron Microscopy

    LRRC4 inhibits GBM cell autophagy activation. a The stably expressed LRRC4 decreased the expression of LC3B in U251 and PG2 cells. Knockdown LRRC4 restored the expression of LC3B in cells that stably expressed LRRC4. Cells were lysed and protein was extracted. LRRC4, LC3B and GAPDH were evaluated by western blotting. Vector: control plasmid, C4: LRRC4 plasmid. siC4-1 and siC4-2 are siRNAs that targeted LRRC4. Numbers represent the relative ratio of LC3II and GAPDH. b U251 cells were transfected with increasing concentrations of LRRC4 plasmid. Cells were lysed and protein was extracted. LRRC4, LC3B, P62 and GAPDH were evaluated by western blotting. LRRC4: LRRC4 plasmid. Numbers represent the relative ratio of LC3II and GAPDH, P62 and GAPDH. c U251 cells were transfected with LRRC4 or control plasmid and cells were cultured in normal and starvation conditions. LRRC4, LC3B, P62 and GAPDH were evaluated by western blotting. Vector: control plasmid, C4: LRRC4 plasmid. Numbers represent the relative ratio of LC3II, P62 and GAPDH. d U251 cells (upper) and PG2 cells (bottom) that stably expressed mRFP-EGFP-LC3 fusion protein were transfected with LRRC4 plasmid. Confocal microscopic analysis is shown. Scale bar, 5 μm. Vector: control plasmid, C4: LRRC4 plasmid. Note: PG2 cells were primary cultured glioblastoma cells. e U251 cells (upper) and PG2 cells (bottom) were transfected with LRRC4 plasmid. Cells were immunostained with an LC3B antibody. Fluorescence microscope analysis is shown. Scale bar, 20 μm. Vector: control plasmid, C4: LRRC4 plasmid. f , g Autophagosomes were observed by transmission electron microscopy in U251 cells and PG2 cells that were transfected with LRRC4 plasmid or control vector. Scale bar, 1 μm. Vector: control plasmid, LRRC4: LRRC4 plasmid.
    Figure Legend Snippet: LRRC4 inhibits GBM cell autophagy activation. a The stably expressed LRRC4 decreased the expression of LC3B in U251 and PG2 cells. Knockdown LRRC4 restored the expression of LC3B in cells that stably expressed LRRC4. Cells were lysed and protein was extracted. LRRC4, LC3B and GAPDH were evaluated by western blotting. Vector: control plasmid, C4: LRRC4 plasmid. siC4-1 and siC4-2 are siRNAs that targeted LRRC4. Numbers represent the relative ratio of LC3II and GAPDH. b U251 cells were transfected with increasing concentrations of LRRC4 plasmid. Cells were lysed and protein was extracted. LRRC4, LC3B, P62 and GAPDH were evaluated by western blotting. LRRC4: LRRC4 plasmid. Numbers represent the relative ratio of LC3II and GAPDH, P62 and GAPDH. c U251 cells were transfected with LRRC4 or control plasmid and cells were cultured in normal and starvation conditions. LRRC4, LC3B, P62 and GAPDH were evaluated by western blotting. Vector: control plasmid, C4: LRRC4 plasmid. Numbers represent the relative ratio of LC3II, P62 and GAPDH. d U251 cells (upper) and PG2 cells (bottom) that stably expressed mRFP-EGFP-LC3 fusion protein were transfected with LRRC4 plasmid. Confocal microscopic analysis is shown. Scale bar, 5 μm. Vector: control plasmid, C4: LRRC4 plasmid. Note: PG2 cells were primary cultured glioblastoma cells. e U251 cells (upper) and PG2 cells (bottom) were transfected with LRRC4 plasmid. Cells were immunostained with an LC3B antibody. Fluorescence microscope analysis is shown. Scale bar, 20 μm. Vector: control plasmid, C4: LRRC4 plasmid. f , g Autophagosomes were observed by transmission electron microscopy in U251 cells and PG2 cells that were transfected with LRRC4 plasmid or control vector. Scale bar, 1 μm. Vector: control plasmid, LRRC4: LRRC4 plasmid.

    Techniques Used: Activation Assay, Stable Transfection, Expressing, Western Blot, Plasmid Preparation, Transfection, Cell Culture, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy

    31) Product Images from "Ginsenoside Rg3 promotes regression from hepatic fibrosis through reducing inflammation-mediated autophagy signaling pathway"

    Article Title: Ginsenoside Rg3 promotes regression from hepatic fibrosis through reducing inflammation-mediated autophagy signaling pathway

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2597-7

    G-Rg3 ameliorated hepatic fibrosis through inhibiting autophagy flux both in vivo and in vitro. a Effect of G-Rg3 on autophagy-related proteins in TAA-Chronic model. The ratio of LC3b/LC3a represented an ignition of autophagy flux. Then, LPS-induced autophagy model was established in HSC-T6 cells. b Experiments for investigation of LPS doses (0–100 ng/mL) by western blot and normalized by GAPDH. c Evaluations of LPS treatment at 0, 2, 4, 8, 12, and 24 h were measured by expressions of α-SMA, LC3 a/b, p62. d Immunofluorescence staining of α-SMA with different concentrations of G-Rg3 incubation with an amplification of ×400. e Time of G-Rg3 treatment at 0, 2, 4, 8, 12, and 24 h were detected by western blot. f Expressions of TGF-β1 and α-SMA in HSC-T6 cells with G-Rg3 treatment. Values were shown as the mean ± S.D; * p
    Figure Legend Snippet: G-Rg3 ameliorated hepatic fibrosis through inhibiting autophagy flux both in vivo and in vitro. a Effect of G-Rg3 on autophagy-related proteins in TAA-Chronic model. The ratio of LC3b/LC3a represented an ignition of autophagy flux. Then, LPS-induced autophagy model was established in HSC-T6 cells. b Experiments for investigation of LPS doses (0–100 ng/mL) by western blot and normalized by GAPDH. c Evaluations of LPS treatment at 0, 2, 4, 8, 12, and 24 h were measured by expressions of α-SMA, LC3 a/b, p62. d Immunofluorescence staining of α-SMA with different concentrations of G-Rg3 incubation with an amplification of ×400. e Time of G-Rg3 treatment at 0, 2, 4, 8, 12, and 24 h were detected by western blot. f Expressions of TGF-β1 and α-SMA in HSC-T6 cells with G-Rg3 treatment. Values were shown as the mean ± S.D; * p

    Techniques Used: In Vivo, In Vitro, Western Blot, Immunofluorescence, Staining, Incubation, Amplification

    32) Product Images from "Gαi2 signaling regulates inflammasome priming and cytokine production by biasing macrophage phenotype determination"

    Article Title: Gαi2 signaling regulates inflammasome priming and cytokine production by biasing macrophage phenotype determination

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1801145

    Gα i2 modulates signaling downstream of LPS and IL-4. BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice were treated with LPS (250 ng/ml) for the indicated time. Whole cell lysates were collected and separated on an SDS PAGE gel. Immunoblots are shown for A) NFκB related protein p-65 (phospho and total) and GAPDH, and B) MAPK cascade proteins ERK1/2 (phospho and total) and JNK1/2 (phospho and total). C) Phosflow analysis of p38 phosphorylation after LPS stimulation (250 ng/ml) for the indicated time in BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice. Data is presented as the mean fluorescence intensity ratio of phospho p38 to total p38 and normalized to the control. D) BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice were treated with LPS (1 μg/ml) for 4 hours, cell culture supernatant was collected and assayed for IFN-β. E) Immunoblotting for STAT3 (phospho and total) and Akt (phospho and total) from lysates obtained from LPS stimulated (250 ng/ml) WT or Gα i2 KO ( Gnai2 −/− ) mice. F) BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice were treated with IL-4 (20 ng/ml) for the indicated time. Immunoblotting for STAT6 (phospho and total) and actin are shown. Phospho protein bands are quantified relative to their respective total protein band. Band intensities are normalized to the indicated control. Experiments were performed a minimum of three times.
    Figure Legend Snippet: Gα i2 modulates signaling downstream of LPS and IL-4. BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice were treated with LPS (250 ng/ml) for the indicated time. Whole cell lysates were collected and separated on an SDS PAGE gel. Immunoblots are shown for A) NFκB related protein p-65 (phospho and total) and GAPDH, and B) MAPK cascade proteins ERK1/2 (phospho and total) and JNK1/2 (phospho and total). C) Phosflow analysis of p38 phosphorylation after LPS stimulation (250 ng/ml) for the indicated time in BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice. Data is presented as the mean fluorescence intensity ratio of phospho p38 to total p38 and normalized to the control. D) BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice were treated with LPS (1 μg/ml) for 4 hours, cell culture supernatant was collected and assayed for IFN-β. E) Immunoblotting for STAT3 (phospho and total) and Akt (phospho and total) from lysates obtained from LPS stimulated (250 ng/ml) WT or Gα i2 KO ( Gnai2 −/− ) mice. F) BMDMs generated from WT and Gα i2 KO ( Gnai2 −/− ) mice were treated with IL-4 (20 ng/ml) for the indicated time. Immunoblotting for STAT6 (phospho and total) and actin are shown. Phospho protein bands are quantified relative to their respective total protein band. Band intensities are normalized to the indicated control. Experiments were performed a minimum of three times.

    Techniques Used: Generated, Mouse Assay, SDS Page, Western Blot, Fluorescence, Cell Culture

    33) Product Images from "Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats"

    Article Title: Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0171491

    Exercise regulated the expressions and activities of Rho GTPases in the cortex and hippocampus in aging rats. (A) Positive and negative controls of Rho GTPasee in pull down assays. (B) Effects of exercise on the expressions and activities of RhoA. (a-b) Immunoreactive bands corresponding to RhoA active and total protein and GAPDH in the cortex (a) and the hippocampus (b). (c-d) Summarized data of RhoA total protein levels (c, as a ratio to GAPDH) and activities (d, as a ratio to total protein) in the cortex and the hippocampus. n = 5 in each group. (C) Effects of exercise on the expressions and activities of Rac1. (a-b) Immunoreactive bands corresponding to Rac1 active and total protein and GAPDH in the cortex (a) and the hippocampus (b). (c-d) Summarized data of Rac1 total protein levels (c, as a ratio to GAPDH) and activities (d, as a ratio to total protein) in the cortex and the hippocampus. n = 5 in each group. (D) Effects of exercise on the expressions and activities of Cdc42. (a-b) Immunoreactive bands corresponding to Cdc42 active and total protein and GAPDH in the cortex (a) and the hippocampus (b). (c-d) Summarized data of Cdc42 total protein levels (c, as a ratio to GAPDH) and activities (d, as a ratio to total protein) in the cortex and the hippocampus. Values are mean and standard error of the mean, n = 5 in each group. *P
    Figure Legend Snippet: Exercise regulated the expressions and activities of Rho GTPases in the cortex and hippocampus in aging rats. (A) Positive and negative controls of Rho GTPasee in pull down assays. (B) Effects of exercise on the expressions and activities of RhoA. (a-b) Immunoreactive bands corresponding to RhoA active and total protein and GAPDH in the cortex (a) and the hippocampus (b). (c-d) Summarized data of RhoA total protein levels (c, as a ratio to GAPDH) and activities (d, as a ratio to total protein) in the cortex and the hippocampus. n = 5 in each group. (C) Effects of exercise on the expressions and activities of Rac1. (a-b) Immunoreactive bands corresponding to Rac1 active and total protein and GAPDH in the cortex (a) and the hippocampus (b). (c-d) Summarized data of Rac1 total protein levels (c, as a ratio to GAPDH) and activities (d, as a ratio to total protein) in the cortex and the hippocampus. n = 5 in each group. (D) Effects of exercise on the expressions and activities of Cdc42. (a-b) Immunoreactive bands corresponding to Cdc42 active and total protein and GAPDH in the cortex (a) and the hippocampus (b). (c-d) Summarized data of Cdc42 total protein levels (c, as a ratio to GAPDH) and activities (d, as a ratio to total protein) in the cortex and the hippocampus. Values are mean and standard error of the mean, n = 5 in each group. *P

    Techniques Used:

    Exercise decreased the increased expression of cofilin in the cortex during aging. (A-B) Immunoreactive bands corresponding to cofilin and GAPDH in the cortex (A) and hippocampus (B). (C-D) Summarized data of cofilin protein levels (as a ratio to GAPDH) in the cortex (C) and hippocampus (D). Values are mean and standard error of the mean, n = 5 in each group. ** P
    Figure Legend Snippet: Exercise decreased the increased expression of cofilin in the cortex during aging. (A-B) Immunoreactive bands corresponding to cofilin and GAPDH in the cortex (A) and hippocampus (B). (C-D) Summarized data of cofilin protein levels (as a ratio to GAPDH) in the cortex (C) and hippocampus (D). Values are mean and standard error of the mean, n = 5 in each group. ** P

    Techniques Used: Expressing

    34) Product Images from "Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis"

    Article Title: Downregulation of HS6ST2 by miR-23b-3p enhances matrix degradation through p38 MAPK pathway in osteoarthritis

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0729-0

    MiR-23b-3p could enhance matrix degradation by means of regulating activity of p38 MAPK in human chondrocytes under TNF-α treatment. a , b SW1353 cells were transfected by mimic miR-23b-3p ( a ) or anti-miR-23b-3p sequence ( b ) with or without TNF-α for 24 h, and the protein expression of phosphorylation form of p38 MAPK (p-p38), total p38 MAPK, and MMP13 was detected by western blotting. Asterisk (*): compared with the mimic NC ( a ) or anti-NC group ( b ). c SW1353 cells were treated with mimic miR-23b-3p with or without p38 MAPK inhibitor SB203580 (10 μM) under stimulation of TNF-α. The protein expression of MMP13 was determined by western blotting. Asterisk (*): compared with the mimic NC group. d Interfering efficiency of siRNAs against p38 MAPK was determined by western blotting under 50 nM p38 siRNA, 50 nM negative control (NC), and Mock (transfection regent only) transfection for 48 h. e Under stimulation with TNF-α, SW1353 cells were transfected by mimic miR-23b-3p under treatment with si- p38 mixture (containing three siRNA target sequences), and p-p38, total p38, and MMP13 were determined by western blotting. Asterisk (*): compared with mimic NC or si-NC. Each relative expression of phosphorylation form was normalized by the total form. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
    Figure Legend Snippet: MiR-23b-3p could enhance matrix degradation by means of regulating activity of p38 MAPK in human chondrocytes under TNF-α treatment. a , b SW1353 cells were transfected by mimic miR-23b-3p ( a ) or anti-miR-23b-3p sequence ( b ) with or without TNF-α for 24 h, and the protein expression of phosphorylation form of p38 MAPK (p-p38), total p38 MAPK, and MMP13 was detected by western blotting. Asterisk (*): compared with the mimic NC ( a ) or anti-NC group ( b ). c SW1353 cells were treated with mimic miR-23b-3p with or without p38 MAPK inhibitor SB203580 (10 μM) under stimulation of TNF-α. The protein expression of MMP13 was determined by western blotting. Asterisk (*): compared with the mimic NC group. d Interfering efficiency of siRNAs against p38 MAPK was determined by western blotting under 50 nM p38 siRNA, 50 nM negative control (NC), and Mock (transfection regent only) transfection for 48 h. e Under stimulation with TNF-α, SW1353 cells were transfected by mimic miR-23b-3p under treatment with si- p38 mixture (containing three siRNA target sequences), and p-p38, total p38, and MMP13 were determined by western blotting. Asterisk (*): compared with mimic NC or si-NC. Each relative expression of phosphorylation form was normalized by the total form. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value

    Techniques Used: Activity Assay, Transfection, Sequencing, Expressing, Western Blot, Negative Control, MANN-WHITNEY

    HS6ST2 could regulate the matrix degradation depending on the activity of p38 MAPK. a SW1353 cells were transfected by si -HS6ST2 or negative control (si-NC) with or without TNF-α for 24 h, and the protein expression of p-p38 and p38 was detected by western blotting. Asterisk (*): compared with the si-NC group. b Under stimulation with TNF-α, SW1353 cells were transfected by empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) under treatment with mimic miR-23b-3p, and p-p38 and total p38 were determined by western blotting. Asterisk (*): compared with the mimic NC group. c SW1353 cells were transfected with HS6ST2 siRNA with or without p38 MAPK inhibitor SB203580 (10 μM) after treatment of TNF-α, and the protein expression of MMP13 was determined by western blotting. Each relative expression of phosphorylation form was normalized by the total form. d Schematic representation of miR-23b-3p–HS6ST2 axis-mediated catabolic effects in human chondrocyte. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
    Figure Legend Snippet: HS6ST2 could regulate the matrix degradation depending on the activity of p38 MAPK. a SW1353 cells were transfected by si -HS6ST2 or negative control (si-NC) with or without TNF-α for 24 h, and the protein expression of p-p38 and p38 was detected by western blotting. Asterisk (*): compared with the si-NC group. b Under stimulation with TNF-α, SW1353 cells were transfected by empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) under treatment with mimic miR-23b-3p, and p-p38 and total p38 were determined by western blotting. Asterisk (*): compared with the mimic NC group. c SW1353 cells were transfected with HS6ST2 siRNA with or without p38 MAPK inhibitor SB203580 (10 μM) after treatment of TNF-α, and the protein expression of MMP13 was determined by western blotting. Each relative expression of phosphorylation form was normalized by the total form. d Schematic representation of miR-23b-3p–HS6ST2 axis-mediated catabolic effects in human chondrocyte. GAPDH was used as internal controls in western blotting detection. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value

    Techniques Used: Activity Assay, Transfection, Negative Control, Expressing, Western Blot, Plasmid Preparation, MANN-WHITNEY

    MiR-23b-3p could enhance matrix degradation in human chondrocytes via regulating HS6ST2. a , b SW1353 cells transfected with 50 nM HS6ST2 siRNA mixture (containing three target sequences, si- HS6ST2 ) or negative control (si-NC) were stimulated by 10 ng/ml TNF-α for 24 h. Protein level of HS6ST2 and MMP13 were assayed by western blotting ( a ) and matrix content of chondrocytes was determined by toluidine blue staining ( b ). Asterisk (*): compared with the si-NC group. c , d SW1353 cells transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for 24 h were stimulated by TNF-α for another 24 h. The protein expression of MMP13 was assayed by western blotting ( c ) and matrix content of chondrocytes was determined by toluidine blue staining ( d ). Asterisk (*): compared with the FLAG-Ctrl group. e , f Under stimulation with TNF-α, SW1353 cells were treated with mimic NC or mimic miR-23b-3p for 24 h and then transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for another 24 h to observe the rescuing effect of mimic miR-23b-3p in MMP13 expression ( e ) and matrix content ( f ). Asterisk (*): compared with the mimic NC group. GAPDH was used as internal controls in western blotting detection. In toluidine blue staining results, scale bar, 100 μm. Lower panel, statistical analysis of average optical density of matrix staining of toluidine blue. Bars represent standard error of the mean (SEM) from three independent experiments. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value
    Figure Legend Snippet: MiR-23b-3p could enhance matrix degradation in human chondrocytes via regulating HS6ST2. a , b SW1353 cells transfected with 50 nM HS6ST2 siRNA mixture (containing three target sequences, si- HS6ST2 ) or negative control (si-NC) were stimulated by 10 ng/ml TNF-α for 24 h. Protein level of HS6ST2 and MMP13 were assayed by western blotting ( a ) and matrix content of chondrocytes was determined by toluidine blue staining ( b ). Asterisk (*): compared with the si-NC group. c , d SW1353 cells transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for 24 h were stimulated by TNF-α for another 24 h. The protein expression of MMP13 was assayed by western blotting ( c ) and matrix content of chondrocytes was determined by toluidine blue staining ( d ). Asterisk (*): compared with the FLAG-Ctrl group. e , f Under stimulation with TNF-α, SW1353 cells were treated with mimic NC or mimic miR-23b-3p for 24 h and then transfected with empty vector (FLAG-Ctrl) or pcDNA3.1-FLAG- HS6ST2 vector (FLAG- HS6ST2 ) for another 24 h to observe the rescuing effect of mimic miR-23b-3p in MMP13 expression ( e ) and matrix content ( f ). Asterisk (*): compared with the mimic NC group. GAPDH was used as internal controls in western blotting detection. In toluidine blue staining results, scale bar, 100 μm. Lower panel, statistical analysis of average optical density of matrix staining of toluidine blue. Bars represent standard error of the mean (SEM) from three independent experiments. Mann–Whitney U test was used to identify statistical differences between two groups. Asterisk (*) or hash ( # ) stands for P value

    Techniques Used: Transfection, Negative Control, Western Blot, Staining, Plasmid Preparation, Expressing, MANN-WHITNEY

    35) Product Images from "Hydrogen Sulfide Alleviates Acute Myocardial Ischemia Injury by Modulating Autophagy and Inflammation Response under Oxidative Stress"

    Article Title: Hydrogen Sulfide Alleviates Acute Myocardial Ischemia Injury by Modulating Autophagy and Inflammation Response under Oxidative Stress

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/3402809

    SOD1 KO mice showed enhanced autophagy levels in ischemic myocardium. (a) Representative blots of LC3II/LC3I, Bax, Bcl2, and S6K phosphorylation and AMPK phosphorylation and quantitative analysis of LC3II/LC3I, Bax, Bcl2, p-S6K, and p-AMPK. (b) Representative images of TEM results are shown. Red arrows indicate autophagic vacuoles. Scale bar = 0.5 μ m. (c) Myocardium LC3 detection with the presence of 40 mg/kg chloroquine. Representative blots and quantitative analysis of LC3II/GAPDH are shown. Values are mean ± SE, n = 6 in each group. ∗ p
    Figure Legend Snippet: SOD1 KO mice showed enhanced autophagy levels in ischemic myocardium. (a) Representative blots of LC3II/LC3I, Bax, Bcl2, and S6K phosphorylation and AMPK phosphorylation and quantitative analysis of LC3II/LC3I, Bax, Bcl2, p-S6K, and p-AMPK. (b) Representative images of TEM results are shown. Red arrows indicate autophagic vacuoles. Scale bar = 0.5 μ m. (c) Myocardium LC3 detection with the presence of 40 mg/kg chloroquine. Representative blots and quantitative analysis of LC3II/GAPDH are shown. Values are mean ± SE, n = 6 in each group. ∗ p

    Techniques Used: Mouse Assay, Transmission Electron Microscopy

    36) Product Images from "Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation"

    Article Title: Lactobacillus rhamnosus GR-1 Ameliorates Escherichia coli-Induced Inflammation and Cell Damage via Attenuation of ASC-Independent NLRP3 Inflammasome Activation

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03044-15

    Western blot detection of NLRP3, ASC, and caspase-1. Representative panels showing expression of NLRP3 (A, left panels), ASC (B, left panels), and caspase-1 (C, left panels) by BMECs collected from the indicated BMEC cultures at 2, 4, and 6 h after E. coli challenge. The intensities of NLRP3, ASC, and caspase-1 bands were determined using Quantity One software. Expression of GAPDH was measured as an internal control. Results are presented as the ratio of NLRP3, ASC, or caspase-1 band intensity to that of GAPDH (right panels). Data are presented as the means ± the SEM of three independent experiments. *, P
    Figure Legend Snippet: Western blot detection of NLRP3, ASC, and caspase-1. Representative panels showing expression of NLRP3 (A, left panels), ASC (B, left panels), and caspase-1 (C, left panels) by BMECs collected from the indicated BMEC cultures at 2, 4, and 6 h after E. coli challenge. The intensities of NLRP3, ASC, and caspase-1 bands were determined using Quantity One software. Expression of GAPDH was measured as an internal control. Results are presented as the ratio of NLRP3, ASC, or caspase-1 band intensity to that of GAPDH (right panels). Data are presented as the means ± the SEM of three independent experiments. *, P

    Techniques Used: Western Blot, Expressing, Software

    37) Product Images from "Pin1 mediates Aβ42-induced dendritic spine loss"

    Article Title: Pin1 mediates Aβ42-induced dendritic spine loss

    Journal: Science signaling

    doi: 10.1126/scisignal.aap8734

    Aβ42 inhibits Pin1 isomerase activity ( A ) Western blotting for Pin1, Snap25, βIII-tubulin and GAPDH in soluble fractions of temporal cortex lysates from 3 Alzheimer’s disease (AD) patients and 3 age-matched control individuals (not diagnosed with dementia; Con). ( B ) Pin1 activity assay in equal amounts of soluble protein extract from control and AD patient brain tissues described in (A). ( C ) Pin1 activity assay with murine synaptoneurosomes (SN) that were untreated (red) or treated for 10 min with control brain extract (+HC, black), AD brain extract (+AD, purple), or Aβ42 (green). ( D ) Pin1 activity assay in SNs that were either untreated (red) or treated with juglone, PiB (, TAT-WW, TAT-W34A, or Aβ42. Data are means of N ≥ 8 replicates per treatment. * P
    Figure Legend Snippet: Aβ42 inhibits Pin1 isomerase activity ( A ) Western blotting for Pin1, Snap25, βIII-tubulin and GAPDH in soluble fractions of temporal cortex lysates from 3 Alzheimer’s disease (AD) patients and 3 age-matched control individuals (not diagnosed with dementia; Con). ( B ) Pin1 activity assay in equal amounts of soluble protein extract from control and AD patient brain tissues described in (A). ( C ) Pin1 activity assay with murine synaptoneurosomes (SN) that were untreated (red) or treated for 10 min with control brain extract (+HC, black), AD brain extract (+AD, purple), or Aβ42 (green). ( D ) Pin1 activity assay in SNs that were either untreated (red) or treated with juglone, PiB (, TAT-WW, TAT-W34A, or Aβ42. Data are means of N ≥ 8 replicates per treatment. * P

    Techniques Used: Activity Assay, Western Blot

    38) Product Images from "High Expression of ITGA3 Promotes Proliferation and Cell Cycle Progression and Indicates Poor Prognosis in Intrahepatic Cholangiocarcinoma"

    Article Title: High Expression of ITGA3 Promotes Proliferation and Cell Cycle Progression and Indicates Poor Prognosis in Intrahepatic Cholangiocarcinoma

    Journal: BioMed Research International

    doi: 10.1155/2018/2352139

    Expression of ITGA3 in ICC cell lines . (a) Western blot analysis and quantitative results for the relative expression of ITGA3 in ICC cell lines relative to GAPDH. (b) Identification of ITGA3 in HuccT-1 cells after transfection with siRNAs for ITGA3. (c) Identification of ITGA3 in Hccc-9810 cells after transfection with siRNAs for ITGA3. Si-1, Si-2, and Si-3 are three different siRNAs that affect different truncations. Abbreviations . ICC: intrahepatic cholangiocarcinoma; ITGA3: integrin subunit alpha 3; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC: negative control; siRNA: short interfering RNA.
    Figure Legend Snippet: Expression of ITGA3 in ICC cell lines . (a) Western blot analysis and quantitative results for the relative expression of ITGA3 in ICC cell lines relative to GAPDH. (b) Identification of ITGA3 in HuccT-1 cells after transfection with siRNAs for ITGA3. (c) Identification of ITGA3 in Hccc-9810 cells after transfection with siRNAs for ITGA3. Si-1, Si-2, and Si-3 are three different siRNAs that affect different truncations. Abbreviations . ICC: intrahepatic cholangiocarcinoma; ITGA3: integrin subunit alpha 3; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; NC: negative control; siRNA: short interfering RNA.

    Techniques Used: Expressing, Immunocytochemistry, Western Blot, Transfection, Negative Control, Small Interfering RNA

    39) Product Images from "Hydrogen Sulfide Alleviates Acute Myocardial Ischemia Injury by Modulating Autophagy and Inflammation Response under Oxidative Stress"

    Article Title: Hydrogen Sulfide Alleviates Acute Myocardial Ischemia Injury by Modulating Autophagy and Inflammation Response under Oxidative Stress

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/3402809

    SOD1 KO mice showed enhanced autophagy levels in ischemic myocardium. (a) Representative blots of LC3II/LC3I, Bax, Bcl2, and S6K phosphorylation and AMPK phosphorylation and quantitative analysis of LC3II/LC3I, Bax, Bcl2, p-S6K, and p-AMPK. (b) Representative images of TEM results are shown. Red arrows indicate autophagic vacuoles. Scale bar = 0.5 μ m. (c) Myocardium LC3 detection with the presence of 40 mg/kg chloroquine. Representative blots and quantitative analysis of LC3II/GAPDH are shown. Values are mean ± SE, n = 6 in each group. ∗ p
    Figure Legend Snippet: SOD1 KO mice showed enhanced autophagy levels in ischemic myocardium. (a) Representative blots of LC3II/LC3I, Bax, Bcl2, and S6K phosphorylation and AMPK phosphorylation and quantitative analysis of LC3II/LC3I, Bax, Bcl2, p-S6K, and p-AMPK. (b) Representative images of TEM results are shown. Red arrows indicate autophagic vacuoles. Scale bar = 0.5 μ m. (c) Myocardium LC3 detection with the presence of 40 mg/kg chloroquine. Representative blots and quantitative analysis of LC3II/GAPDH are shown. Values are mean ± SE, n = 6 in each group. ∗ p

    Techniques Used: Mouse Assay, Transmission Electron Microscopy

    40) Product Images from "Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice"

    Article Title: Microtubule associated protein 4 phosphorylation leads to pathological cardiac remodeling in mice

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.10.017

    MAP4 phosphorylation induces MT disassembly and mitochondrial translocation of p-MAP4. (a, b) WB analysis was used to detect polymerized (poly) or free tubulin in two mouse models. VDAC and GAPDH were used as the internal controls for poly and free tubulin, respectively. n = 6. (c) Determination of MAP4 binding to tubulin in two mouse models by immunoprecipitation (IP). n = 6. (d, e) WB analysis showing the levels of p-M in mitochondria fractions and fractions apart from mitochondria in two mouse models. n = 6. (f, g) Poly/free tubulin in CMs were detected by WB. n = 5. The experiment was conducted 5 times. (h) Representative confocal immunofluorescence images showing the MTs organization of the CMs. Bar, 10 μm. The experiment was conducted 5 times. The inserts showed high magnification images of the representative MT network. (i, j) Detection of p-M translocation to mitochondria with or without MAP4(Ala) transfection in CMs. n = 5. The experiment was conducted 5 times. The Data were shown as mean ± SEM. *P
    Figure Legend Snippet: MAP4 phosphorylation induces MT disassembly and mitochondrial translocation of p-MAP4. (a, b) WB analysis was used to detect polymerized (poly) or free tubulin in two mouse models. VDAC and GAPDH were used as the internal controls for poly and free tubulin, respectively. n = 6. (c) Determination of MAP4 binding to tubulin in two mouse models by immunoprecipitation (IP). n = 6. (d, e) WB analysis showing the levels of p-M in mitochondria fractions and fractions apart from mitochondria in two mouse models. n = 6. (f, g) Poly/free tubulin in CMs were detected by WB. n = 5. The experiment was conducted 5 times. (h) Representative confocal immunofluorescence images showing the MTs organization of the CMs. Bar, 10 μm. The experiment was conducted 5 times. The inserts showed high magnification images of the representative MT network. (i, j) Detection of p-M translocation to mitochondria with or without MAP4(Ala) transfection in CMs. n = 5. The experiment was conducted 5 times. The Data were shown as mean ± SEM. *P

    Techniques Used: Translocation Assay, Western Blot, Binding Assay, Immunoprecipitation, Immunofluorescence, Transfection

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    Article Snippet: .. After blocking with TBST (0.1% Tween-20) containing 5% skimmed milk at room temperature for 2 h, the membranes were then incubated with primary antibodies (1:1,000) against WNT2B (cat. no. ab50575; Abcam), β-catenin (cat. no. 51067-2-AP; Proteintech), TCF4 (cat. no. 22337-1-AP; Proteintech), N-cadherin (cat. no. 22018-1-AP; Proteintech), vimentin (cat. no. 10366-1-AP; Proteintech), cyclin D1 (cat. no. 26939-1-AP; Proteintech), CDK2 (cat. no. 10122-1-AP; Proteintech), c-Myc (cat. no. 10828-1-AP; Proteintech) and GAPDH (cat. no. 60004-1-Ig; Proteintech) for 16 h at 4°C. .. After washing with TBST to remove unbound antibodies, the membranes were incubated with the horseradish peroxidase conjugated secondary antibodies goat anti-rabbit (1:3,000; cat. no. SA00001-2, Proteintech) and goat anti-mouse (1:3,000; cat. no. SA00001-1, Proteintech) for 2 h at room temperature.

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    Proteintech anti gapdh
    Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and <t>GAPDH)</t> in exosomes. <t>FASN</t> and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS
    Anti Gapdh, supplied by Proteintech, used in various techniques. Bioz Stars score: 99/100, based on 413 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and GAPDH) in exosomes. FASN and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS

    Journal: Journal of Ovarian Research

    Article Title: Proteomic and lipidomic analysis of exosomes derived from ovarian cancer cells and ovarian surface epithelial cells

    doi: 10.1186/s13048-020-0609-y

    Figure Lengend Snippet: Validation of the MS/MS results by Western blotting. Western blot analyses show increased intensities for exosomal marker (TSG101) and lower intensities for negative control markers (β-Actin, β-tubulin and GAPDH) in exosomes. FASN and L1CAM was only verified in exosomes derived from HOSEPiC as identified by MS/MS

    Article Snippet: The antibodies used were anti-FASN (ABclonal, A0462), anti-L1CAM (ABclonal, A8555), anti-TSG101 (Proteintech, 14,497–1-AP), anti-GAPDH (Proteintech, 60,004–1-Ig), and anti-β-Actin (Proteintech, 60,008–1-Ig).

    Techniques: Tandem Mass Spectroscopy, Western Blot, Marker, Negative Control, Derivative Assay

    miR-137 represses cholangiocarcinoma cell proliferation in vitro . (A) Fluorescence microscope examination (magnification, ×200) and reverse transcription-quantitative PCR were used to detect the infection efficiency of miR-137 overexpression (LV-miR-137) and NC lentiviruses. (B) The effect of miR-137 on cholangiocarcinoma cell proliferation was detected by the Cell Counting Kit-8 assay. (C) The effect of miR-137 on cholangiocarcinoma cell colony formation ability was detected using colony formation assays. (D) Cell cycle distribution was analyzed after miR-137 overexpression in TFK-1 and HuCCT1 cells. (E) Western blotting was used to detect the expression of CDK2 and cyclin D1 in the miR-137 overexpression and normal control groups. GAPDH was used as the loading control. * P

    Journal: International Journal of Molecular Medicine

    Article Title: MicroRNA-137 suppresses the proliferation, migration and invasion of cholangiocarcinoma cells by targeting WNT2B

    doi: 10.3892/ijmm.2020.4474

    Figure Lengend Snippet: miR-137 represses cholangiocarcinoma cell proliferation in vitro . (A) Fluorescence microscope examination (magnification, ×200) and reverse transcription-quantitative PCR were used to detect the infection efficiency of miR-137 overexpression (LV-miR-137) and NC lentiviruses. (B) The effect of miR-137 on cholangiocarcinoma cell proliferation was detected by the Cell Counting Kit-8 assay. (C) The effect of miR-137 on cholangiocarcinoma cell colony formation ability was detected using colony formation assays. (D) Cell cycle distribution was analyzed after miR-137 overexpression in TFK-1 and HuCCT1 cells. (E) Western blotting was used to detect the expression of CDK2 and cyclin D1 in the miR-137 overexpression and normal control groups. GAPDH was used as the loading control. * P

    Article Snippet: After blocking with TBST (0.1% Tween-20) containing 5% skimmed milk at room temperature for 2 h, the membranes were then incubated with primary antibodies (1:1,000) against WNT2B (cat. no. ab50575; Abcam), β-catenin (cat. no. 51067-2-AP; Proteintech), TCF4 (cat. no. 22337-1-AP; Proteintech), N-cadherin (cat. no. 22018-1-AP; Proteintech), vimentin (cat. no. 10366-1-AP; Proteintech), cyclin D1 (cat. no. 26939-1-AP; Proteintech), CDK2 (cat. no. 10122-1-AP; Proteintech), c-Myc (cat. no. 10828-1-AP; Proteintech) and GAPDH (cat. no. 60004-1-Ig; Proteintech) for 16 h at 4°C.

    Techniques: In Vitro, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Infection, Over Expression, Cell Counting, Western Blot, Expressing

    The p53 stabilizer nutlin‐3 induced MMT through ROS generation but not through the p53 transcription/mitochondria‐dependent signaling pathway. A, RNA‐seq analysis of monocytes obtained from two healthy donors (D72 and D73), and treated or not treated with 100 µM H 2 O 2 for 7 days. The heatmap shows hierarchical clustering of mRNA levels of genes in monocytes treated as indicated; the volcano plot shows differentially expressed genes plotted as the log2(fold change) versus the –log10( P ‐value). A total of 94 differentially expressed genes between the H 2 O 2 ‐treated cell group and the control cell group exceeded the established thresholds (–log10( P ‐value) > 1.30 and |log2‐fold change| > .585; upregulated genes are shown in red, and downregulated genes are shown in green). KEGG pathway analysis was performed on the upregulated genes identified by RNA‐seq. B and C, Western blot analysis of the expression of p53 and the target genes p21 and MDM2 in freshly isolated monocytes (0 day), monocytes treated with 0/50/100 µM H 2 O 2 for 3/7 days, and monocytes treated with radiation (0/4/8 Gy) for 3 days. GAPDH was used as the loading control. D and E, Western blot analysis of the expression of αSMA, p53, p21, and MDM2 in monocytes treated or not treated with nutlin‐3, H 2 O 2 , PFT‐α, or PFT‐μ. GAPDH was used as the loading control. F, Representative flow cytometric analysis of intracellular ROS levels in monocytes treated or not treated with nutin‐3 (10 µM) or the same volume of DMSO (blue, ctrl; green, DMSO; red, nutlin‐3). G, Western blot analysis of monocytes treated or not treated with 10 µM nutlin‐3 or 100U/mL catalase. GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; RNA‐seq, RNA sequencing; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMSO, dimethyl sulfoxide.

    Journal: Clinical and Translational Medicine

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    doi: 10.1002/ctm2.41

    Figure Lengend Snippet: The p53 stabilizer nutlin‐3 induced MMT through ROS generation but not through the p53 transcription/mitochondria‐dependent signaling pathway. A, RNA‐seq analysis of monocytes obtained from two healthy donors (D72 and D73), and treated or not treated with 100 µM H 2 O 2 for 7 days. The heatmap shows hierarchical clustering of mRNA levels of genes in monocytes treated as indicated; the volcano plot shows differentially expressed genes plotted as the log2(fold change) versus the –log10( P ‐value). A total of 94 differentially expressed genes between the H 2 O 2 ‐treated cell group and the control cell group exceeded the established thresholds (–log10( P ‐value) > 1.30 and |log2‐fold change| > .585; upregulated genes are shown in red, and downregulated genes are shown in green). KEGG pathway analysis was performed on the upregulated genes identified by RNA‐seq. B and C, Western blot analysis of the expression of p53 and the target genes p21 and MDM2 in freshly isolated monocytes (0 day), monocytes treated with 0/50/100 µM H 2 O 2 for 3/7 days, and monocytes treated with radiation (0/4/8 Gy) for 3 days. GAPDH was used as the loading control. D and E, Western blot analysis of the expression of αSMA, p53, p21, and MDM2 in monocytes treated or not treated with nutlin‐3, H 2 O 2 , PFT‐α, or PFT‐μ. GAPDH was used as the loading control. F, Representative flow cytometric analysis of intracellular ROS levels in monocytes treated or not treated with nutin‐3 (10 µM) or the same volume of DMSO (blue, ctrl; green, DMSO; red, nutlin‐3). G, Western blot analysis of monocytes treated or not treated with 10 µM nutlin‐3 or 100U/mL catalase. GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; RNA‐seq, RNA sequencing; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; DMSO, dimethyl sulfoxide.

    Article Snippet: 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

    Techniques: RNA Sequencing Assay, Western Blot, Expressing, Isolation

    ROS induced MMT through p38‐MAPK signaling pathway. A‐C, Western blot analysis of p‐p38 and t‐p38 expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) at different time points (≤30 min). GAPDH was used as the loading control. D, Western blot analysis of αSMA expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) and the p38 inhibitor SB203580 (0/0.5/1/2 µM). GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MAPK, mitogen‐activated protein kinase.

    Journal: Clinical and Translational Medicine

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    doi: 10.1002/ctm2.41

    Figure Lengend Snippet: ROS induced MMT through p38‐MAPK signaling pathway. A‐C, Western blot analysis of p‐p38 and t‐p38 expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) at different time points (≤30 min). GAPDH was used as the loading control. D, Western blot analysis of αSMA expression in monocytes treated with H 2 O 2 (100 µM), nutlin‐3 (10 µM), or radiation (8 Gy) and the p38 inhibitor SB203580 (0/0.5/1/2 µM). GAPDH was used as the loading control. Data are representative of three experiments Abbreviations: MAPK, mitogen‐activated protein kinase.

    Article Snippet: 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

    Techniques: Western Blot, Expressing

    Oxidative stress induced MMT in vitro. A, Western blot analysis of fibronectin, CD68, and αSMA expression (3 days). GAPDH was used as the loading control. B, Representative photograph of human monocyte‐derived macrophages cultured in vitro (7 days). C, Representative immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in monocytes cultured for 3 or 7 days in vitro. D, A CCK‐8 assay was used to evaluate the cytotoxicity of H 2 O 2 to monocytes cultured in vitro for 1 or 7 days. Data from one representative donor of three donors are shown. Two‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the experimental and ctrl groups. E, Western blot analysis of αSMA, vimentin, FSP1, and CD68 expression. GAPDH was used as the loading control. F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) measurement of αSMA gene expression levels. GAPDH was used as the loading control. One‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the groups. G, Immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in macrophages treated or not with treated 100 µM H 2 O 2 . Cells treated with H 2 O 2 tended to be spindle shaped. H, ELISA of fibronectin concentrations in supernatants from monocytes treated or not treated with 100 µM H 2 O 2 (n = 5). A paired t ‐test was used for comparisons. I, RT‐qPCR measurement of fibronectin gene expression levels (n = 4). The Wilcoxon matched‐pairs test was used for comparison. J, Western blot analysis of fibronectin, αSMA, and CD68 expression in monocytes treated with H 2 O 2 (100 µM, ±) or the H 2 O 2 ‐scavenging enzyme catalase (100 U/mL, ±) on day 3. GAPDH was used as the loading control. K, Flow cytometric analysis of intracellular ROS levels in monocytes irradiated with 8 Gy or sham‐irradiated, using H2DCFDA fluorescent probe (green, without probe; blue, sham‐irradiated; red, irradiated with 8 Gy). A paired t ‐test was used for comparisons between Ctrl and 8 Gy groups (n = 3). L, Western blot analysis of αSMA expression on day 3 after irradiation (0/4/8 Gy). GAPDH was used as the loading control. The data are expressed as the means ± standard errors of the mean and are representative of at least three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; NFs, normal fibroblasts; CAFs, cancer associated fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; FSP1, fibroblast‐specific protein 1; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; ns, not significant ( P > .05). * P

    Journal: Clinical and Translational Medicine

    Article Title: Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma, et al. Oxidative stress induces monocyte‐to‐myofibroblast transdifferentiation through p38 in pancreatic ductal adenocarcinoma

    doi: 10.1002/ctm2.41

    Figure Lengend Snippet: Oxidative stress induced MMT in vitro. A, Western blot analysis of fibronectin, CD68, and αSMA expression (3 days). GAPDH was used as the loading control. B, Representative photograph of human monocyte‐derived macrophages cultured in vitro (7 days). C, Representative immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in monocytes cultured for 3 or 7 days in vitro. D, A CCK‐8 assay was used to evaluate the cytotoxicity of H 2 O 2 to monocytes cultured in vitro for 1 or 7 days. Data from one representative donor of three donors are shown. Two‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the experimental and ctrl groups. E, Western blot analysis of αSMA, vimentin, FSP1, and CD68 expression. GAPDH was used as the loading control. F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) measurement of αSMA gene expression levels. GAPDH was used as the loading control. One‐way ANOVA followed by Bonferroni's post hoc test was used to evaluate the significance of the differences between the groups. G, Immunofluorescence staining for αSMA (green), CD68 (red), and DAPI (blue, for nuclear staining) in macrophages treated or not with treated 100 µM H 2 O 2 . Cells treated with H 2 O 2 tended to be spindle shaped. H, ELISA of fibronectin concentrations in supernatants from monocytes treated or not treated with 100 µM H 2 O 2 (n = 5). A paired t ‐test was used for comparisons. I, RT‐qPCR measurement of fibronectin gene expression levels (n = 4). The Wilcoxon matched‐pairs test was used for comparison. J, Western blot analysis of fibronectin, αSMA, and CD68 expression in monocytes treated with H 2 O 2 (100 µM, ±) or the H 2 O 2 ‐scavenging enzyme catalase (100 U/mL, ±) on day 3. GAPDH was used as the loading control. K, Flow cytometric analysis of intracellular ROS levels in monocytes irradiated with 8 Gy or sham‐irradiated, using H2DCFDA fluorescent probe (green, without probe; blue, sham‐irradiated; red, irradiated with 8 Gy). A paired t ‐test was used for comparisons between Ctrl and 8 Gy groups (n = 3). L, Western blot analysis of αSMA expression on day 3 after irradiation (0/4/8 Gy). GAPDH was used as the loading control. The data are expressed as the means ± standard errors of the mean and are representative of at least three experiments Abbreviations: MMT, monocyte‐to‐myofibroblast transdifferentiation; NFs, normal fibroblasts; CAFs, cancer associated fibroblasts; DAPI, 4′,6‐diamidino‐2‐phenylindole; FSP1, fibroblast‐specific protein 1; RT‐qPCR, reverse transcription‐quantitative polymerase chain reaction; ELISA, enzyme‐linked immunosorbent assay; ns, not significant ( P > .05). * P

    Article Snippet: 2.7 Western blotWe performed western blot (WB) analysis as previously described using an ECL kit (4AW011; purchased from 4A Biotech Co., Ltd) and the following antibodies: anti‐GAPDH (1:5000, 60004‐1‐Ig; Proteintech, USA), anti‐αSMA (1:1000, ab124964; Abcam, UK), anti‐fibronectin (1:1000, ab45688; Abcam, UK), anti‐MDM2 (1:1000, #86934; Cell Signaling Technology, USA), anti‐t‐p53 (1:1000, 10442‐1‐AP; Proteintech, USA), anti‐Col1 (1:1000, #84336; Cell Signaling Technology, USA), anti‐vimentin (1:1000, #5741; Cell Signaling Technology, USA), anti‐CD68 (1:5000, 25747‐1‐AP; Proteintech, USA), anti‐p‐p38 (1:1000, #4511; Cell Signaling Technology, USA), anti‐t‐p38 (1:1000, #8690; Cell Signaling Technology, USA), anti‐p21 (1:1000, ab109520; Abcam, UK), and nti‐FSP1 (1:1000, ab124805; Abcam, UK).

    Techniques: In Vitro, Western Blot, Expressing, Derivative Assay, Cell Culture, Immunofluorescence, Staining, CCK-8 Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Irradiation

    PALD1 accumulates in primary cilia of selected cell types upon Hh pathway activation. (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except for for PALD1 −/− where n = 1). Mean values are indicated by horizontal lines. (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation whereas SMO does. 3T3 cells expressing YFP SMO ( Rohatgi et al., 2009 ) were serum-starved and treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 μm. (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1 −/− cells (see Fig. S4 ). (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) ( Li et al., 2014 ). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors (modified from ( Carvalho-Santos et al., 2010 )). Branch color code: purple, opisthokonts; blue, amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, excavates. When present in the respective organism, motile cilia are shown in green and primary cilia in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs are found (black, 100%; dark grey

    Journal: bioRxiv

    Article Title: Time-resolved proteomic profiling of the ciliary Hedgehog response reveals that GPR161 and PKA undergo regulated co-exit from cilia

    doi: 10.1101/2020.07.29.225797

    Figure Lengend Snippet: PALD1 accumulates in primary cilia of selected cell types upon Hh pathway activation. (A) Cell lysates of indicated cell lines were separated by SDS-PAGE and analyzed by quantitative Western blotting using anti-PALD1 antibody and anti-GAPDH as loading control. Dot plot indicates PALD1 protein levels relative to GAPDH in presence or absence of SAG as indicated (n = 2 except for for PALD1 −/− where n = 1). Mean values are indicated by horizontal lines. (B) PALD1 does not detectably accumulate in primary cilia of 3T3 cells after Hh pathway activation whereas SMO does. 3T3 cells expressing YFP SMO ( Rohatgi et al., 2009 ) were serum-starved and treated with or without SAG for 24 h and analyzed by immunofluorescence microscopy using indicated antibodies. SMO was detected by YFP fluorescence. Scale bars = 2 μm. (C and D) PALD1 is enriched in C2C12 myoblast primary cilia after Hh pathway activation. C2C12 cells were treated and analyzed as in (B). Box plots show background-corrected, relative fluorescence normalized to acetylated tubulin signals. 30 cilia were analyzed for each condition (n = 30). (E) Schematic representation of PALD1 protein. Predicted protein domains and post-translational modifications. Numbers indicate amino acid positions in Mus musculus PALD1, Myr depicts myristoylation site at the N-terminus. Red arrow indicates location of missense mutation in PALD1 −/− cells (see Fig. S4 ). (F) Phylogenetic analysis of PALD1 orthologs and co-conserved proteins (IFT25, CFAP54, Vash1/2, OFD1, CEP19, Phospho1/2) identified by Clustering by Inferred Models of Evolution (CLIME) ( Li et al., 2014 ). The strongest co-conservation with PALD1 was observed for IFT25 (a mobile subunit of the IFT-B complex). Shown is a simplified taxonomic tree with crown eukaryotic groups in different colors (modified from ( Carvalho-Santos et al., 2010 )). Branch color code: purple, opisthokonts; blue, amebozoa; green, plants; yellow, alveolates and heterokonts; orange, haptophytes; and brown, excavates. When present in the respective organism, motile cilia are shown in green and primary cilia in blue. The presence of cilia in T. pseudomonas remains controversial. The presence of the corresponding proteins is indicated by black circles. Conservation of IFT-B complex subunits are depicted by circles with shades of grey that correspond to percentage of subunits, for which orthologs are found (black, 100%; dark grey

    Article Snippet: Antibodies and reagentsAntibodies against the following proteins were used at indicated dilutions: anti-acTub (Sigma-Aldrich T7451, mouse 1:2,000), anti-Arl13b (Proteintech 17711-1-AP, rabbit 1:2,000), anti-IFT88 (Proteintech 13967-1-AP, rabbit 1:200 in IF, 1:1,000 in WB), anti-GFP (raised against 6His-tagged eGFP, rabbit 1:1,000), anti-GPR161 (gift from S. Mukhopadhyay, rabbit 1:500), anti-GLI3 (R & D Systems nachuryAF3690, goat 1:1,000) anti-actin (self-made, rabbit 1:5,000), anti-GAPDH (Proteintech 60004-1-Ig, mouse 1:2,000), anti-PALD1 (Sigma-Aldrich HPA017343, rabbit, IF: 1:250, WB: 1:1,000), anti-SMO (Santa Cruz sc-166685, mouse IgG2a in IF 1:200; Abcam ab236465 for and ), anti-PTCH1 (Abcam ab53715, rabbit, WB 1:1,000), anti-ninein (gift from M. Bornens, rabbit 1:10,000), anti-CEP164 (gift from C. Morison, rabbit, IF 1:2,000), γ-tubulin (Proteintech 66320-1-AP, rabbit 1:1,000).

    Techniques: Activation Assay, SDS Page, Western Blot, Expressing, Immunofluorescence, Microscopy, Fluorescence, Mutagenesis, Modification