Journal: The Journal of Cell Biology

Article Title: Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

doi: 10.1083/jcb.201111063

Figure Lengend Snippet: Bnip3 cooperates with AIF to induce apoptosis and cavitation. (A) 4-d AIF y/+ and AIF y/− EBs were analyzed by immunoblotting for AIF. Actin was used as a loading control. (B) EBs were cultured for 1–4 d and analyzed by immunoblotting for cleaved caspase-3 (cas-3) and actin. Ablation of AIF inhibited caspase-3 activation. (C) Live-phase micrographs show cavitation delay in AIF y/− EBs cultured for 4, 5, and 7 d. After 10 d, most of the AIF y/− EBs were cavitated similar to AIF y/+ EBs. Bars, 100 µm. (D) 4-d EBs were immunostained for cleaved caspase-3. F-actin was stained with rhodamine-phalloidin to show the apical actin belt. Ablation of AIF inhibited apoptosis of the core cells. 5-d EBs were immunostained for the apical marker MUPP1. Apical polarization of the AIF y/− epiblast was not affected despite delayed lumen clearance. (E) AIF y/− ES cells were stably transfected with Bnip3 shRNA (Bnip3 knockdown [KD]) or GFP. 5-d EBs were analyzed by immunoblotting for Bnip3 and cleaved caspase-3. Bnip3 silencing in AIF y/− EBs further inhibited caspase-3 activation. (F) AIF y/+ EBs expressing GFP and AIF y/− EBs stably transfected with Bnip3 shRNA or GFP were cultured for 4, 5, and 7 d. EB cavitation was quantitated by phase microscopy. EB cavitation was significantly delayed in the absence of AIF. Knockdown of Bnip3 in AIF y/− EBs nearly blocked cavitation. n = 6 independent experiments with a total of 529–808 EBs counted for each group. Error bars represent the mean ± SD. *, P < 0.01 versus AIF y/+ GFP; # , P < 0.01 versus AIF y/− GFP.

Article Snippet: Four pGFP-V-RS–based shRNA vectors targeting to mouse Bnip3 and four pRFP-V-RS–based vectors targeting to Bim together with scrambled controls were purchased from OriGene.

Techniques: Western Blot, Cell Culture, Activation Assay, Staining, Marker, Stable Transfection, Transfection, shRNA, Expressing, Microscopy

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. , B. Kaplan-Meier survival curves for wild-type and Usp54-deficient males (A) and females (B). C. TaqMan-based qRT-PCR analysis of Usp54 in MEFs and liver tissues from Usp54 +/+ and Usp54 KF/KF mice. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (**, P < 0.01). D. Body weight curves of Usp54 +/+ and Usp54 KF/KF female mice kept on standard diet. E. Body weight curves of Usp54 +/+ and Usp54 KF/KF female mice kept on high-fat diet and a representative image of females of each genotype at the end of the experiment. F. Total weight gain in the same animals. G. Percentage of gonadal and subscapular fat mass with respect to total body weight of the same animals. H. Mean adipocyte area in gonadal and skin fat. I. Representative histological images. Scale bar: 20 μm (gonadal fat) and 200 μm (skin fat). J. Average thickness of the subcutaneous fat deposits for each genotype. Statistical significance was assessed by a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Oncotarget

Article Title: The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis

doi: 10.18632/oncotarget.12769

Figure Lengend Snippet: A. TaqMan-based qRT-PCR analysis of Usp54 expression in B16F10 cells transduced with the indicated Usp54-specific shRNA or the empty lentiviral vector (pLKO.1) as a control. Data are represented as relative quantification, RQ ± SEM, two-tailed Student's t-test (***, P < 0.001). B. Number of metastases bigger than 200 μm in diameter. Statistical significance was assessed using a non-parametric Mann Whitney-Wilcoxon test (*, P < 0.05; ***, P < 0.001). C. Representative images of lungs and histological analysis for each condition. Scale bar: 200 μm.

Article Snippet: Then, qRT-PCR was performed using TaqMan ® gene expression assay for murine samples ( Usp54 , Mm00513373_m1) or Power SYBR ® Green PCR Master Mix for human cells (Life Technologies), using an Applied Biosystems 7300HT Real-Time PCR System.

Techniques: Quantitative RT-PCR, Expressing, Transduction, shRNA, Plasmid Preparation, Two Tailed Test, MANN-WHITNEY

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: Identification of CD74 as a putative receptor for IBDV. (A) Affinity purification with mAb against the IBDV major capsid protein VP2 in DT40 cells, incubated with VP2 (or the empty vector as a negative control), followed by mass spectrometry analysis. Many host proteins were associated with VP2, including chicken CD74. (B and C) Interaction between VP2 and the CD74 extracellular domain detected via a coimmunoprecipitation (co-IP) assay. (B) Western blot (WB) analysis using an antibody against the HA tag showing the bands corresponding to VP2 in the Flag co-IP assay. (C) Western blot analysis using an antibody against the HA tag showing the bands corresponding to the CD74 extracellular domain in the Flag co-IP assay. (D) To determine the distribution of CD74 in organs, the heart, liver, spleen, lung, kidney, thymus, and bursa of Fabricius of uninfected SPF chickens were collected to measure CD74 transcription levels. The most abundant CD74 expression was found in the bursa. (E) To confirm the involvement of chicken CD74 in the IBDV infection process, SPF chickens were challenged by vvIBDV or PBS. Bursas were collected for RT-qPCR analysis of CD74 transcription levels. CD74 mRNAs were significantly induced by vvIBDV at 12 h p.i. (*, P < 0.05; **, P < 0.01). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Affinity Purification, Incubation, Plasmid Preparation, Negative Control, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Expressing, Infection, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 knockdown suppresses IBDV replication. The expression of the CD74 Ii-2 isoform was downregulated by siRNA interference or knockdown by shRNA in DT40 cells. The vvIBDV Gx strain at an MOI of 1 was added to the CD74 siRNA interference groups or the CD74 KD cell line. Infected cells were washed with PBS at 4 h p.i., and DT40 complete medium was then added. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and qPCR and at 72 h p.i. for ELD50 analysis. (A) CD74 expression levels in DT40 cells determined by Western blotting, showing that CD74 downregulation was effective. siSc., scrambled control siRNA. (B) Western blot assays showing IBDV VP2 expression declining at 24 to 48 h p.i. (C) qPCR analysis showing IBDV copy numbers dropping off at 48 h p.i. (6.34-fold decrease compared with the siRNA negative control; *, P < 0.05). (D) ELD50 assay showing the IBDV titer being downregulated at 48 h p.i. (14.0-fold decrease compared with the siRNA negative control; *, P < 0.05). (E) CD74 mRNA level in DT40 cells determined by qPCR, indicating that CD74 knockdown was effective. (F) IBDV VP2 protein expression was downregulated significantly at 24 to 72 h p.i. in the CD74 KD groups. (G) The IBDV copy number was downregulated at 24 to 72 h p.i. (103- to 104-fold decrease compared with the wild-type [WT] control; P < 0.05). (F) ELD50 assay showing the IBDV titer being downregulated at 72 h p.i. (4.42 × 104-fold decrease compared with wild-type cells; P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Knockdown, Expressing, shRNA, Infection, Western Blot, Control, Negative Control

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 overexpression promotes IBDV replication. CD74 Ii-2 isoform overexpression promotes IBDV infectivity. The expression of the CD74 Ii-2 isoform was upregulated by plasmid transfection in DT40 cells. Next, vvIBDV at an MOI of 1 was used to infected the cells as described in the text. Cells and supernatants were collected at 24, 48, and 72 h p.i. for Western blotting and at 48 h p.i. for qPCR analysis. (A) CD74 Ii-2 isoform overexpression remarkably promotes IBDV replication. The IBDV copy number was increased in the overexpression groups at 72 h p.i. compared with the nonoverexpression group (2.90-fold increase; *, P < 0.05). (B) IBDV VP2 protein expression is upregulated at 48 to 72 h p.i. in the overexpression group.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Over Expression, Infection, Expressing, Plasmid Preparation, Transfection, Western Blot

Journal: Journal of Virology

Article Title: Identification of Chicken CD74 as a Novel Cellular Attachment Receptor for Infectious Bursal Disease Virus in Bursa B Lymphocytes

doi: 10.1128/JVI.01712-19

Figure Lengend Snippet: CD74 isoform Ii-2 confers to vvIBDV the ability to attach to a vvIBDV-nonpermissive cell line. (A) 293T cells (nonpermissive to vvIBDV) were transfected with a full-length chicken CD74 Ii-2 plasmid with an HA tag (or the empty vector as a negative control). Twenty-four hours after transfection, cells were incubated with vvIBDV at an MOI of 5 at 37°C for 36 h to investigate whether CD74 confers susceptibility to IBDV infection. (Top) Cells were processed for confocal analysis, using IBDV VP2 mAb and an HA tag antibody as the primary antibodies. No virus (green fluorescence) was detected in the empty vector control. (Bottom) In the CD74 Ii-2 overexpression group, CD74 (red fluorescence) accumulated on the cell membrane, colocalizing with the IBDV particles (green fluorescence). No virus was observed to enter CD74-overexpressing nonpermissive cells. CD74 could confer attachment ability but not susceptibility to IBDV infection. (B) 293T cells overexpressed HA-tagged chicken CD74 Ii-2 or the empty vector for 24 h and were then incubated with 200 μg SVPs at 4°C for 1 h. After washing with PBS 5 times, cells were processed for confocal analysis as described above. SVPs (green fluorescence) were bound to the membranes of CD74-overexpressing cells (red fluorescence) and colocalized with CD74, but no binding was observed for the empty vector group. (C and D) 293T and HeLa cells (both of which are nonpermissive to vvIBDV) were transfected with a eukaryotic expression plasmid of HA-tagged chicken CD74 Ii-2 (or the empty vector as a negative control) and maintained for 24 h under normal culture conditions. The cells were then incubated with vvIBDV at an MOI of 50 at 4°C for 1 h for the binding assay and washed with PBS 5 times to remove the unbound viruses. Cells were processed for confocal analysis using the same antibodies and procedure as the ones described above. No virus (green fluorescence) was found to bind or infect 293T (C) or HeLa (D) cells transfected with the empty vector. In contrast, IBDV (green fluorescence) colocalized with overexpressed chicken CD74 (red fluorescence) on the membrane of nonpermissive 293T (C) and HeLa (D) cells. (E and F) qPCR analysis indicating that CD74 overexpression promotes IBDV binding to 293T or HeLa cells compared with the empty vector transfection group (9.55-fold increase in 293T cells [E] and 5.07-fold increase in HeLa cells [F]; both P < 0.05). The arithmetic means and standard deviations for at least three independent experiments performed in duplicate are shown.

Article Snippet: Total RNA was extracted from tissues or cells using the RNeasy minikit (Qiagen, Germany), and 1 μg RNA was reverse transcribed to cDNA using the ReverTra Ace qPCR RT master mix with a genomic DNA (gDNA) remover (Toyobo, Japan) in a 20-μl reaction mixture.

Techniques: Transfection, Plasmid Preparation, Negative Control, Incubation, Infection, Virus, Fluorescence, Control, Over Expression, Membrane, Binding Assay, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Differentiation induced by treatment with the Gsk3 inhibitor in hPSCs is β-catenin dependent. (A) H9-7TGP cells were treated with 12 μM CH in mTeSR1 for 4 d. Immunofluorescent staining for Oct4, Isl1, and Nkx2.5 was compared with GFP expression. (Scale bars, 50 μm.) (B and C) 19-9-11 shcat-2 and scramble cells were cultured on Matrigel with mTeSR1 medium containing 12 μM CH for 4 d. (B) RT-PCR analysis of pluripotent, mesendoderm, early mesoderm, and early cardiac gene expression was performed. (C) Oct4 expression on day 4 was analyzed by flow cytometry. Each colored line represents an independent replicate. n = 3. (D) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing CH. After 2 d, the expression of T in the scramble relative to its expression in the shcat-2 line was quantified by quantitative PCR. (E) Flow cytometry analysis of brachyury expression in 19-9-11 shcat-2 and scramble cells exposed to CH for 4 d. Error bars represent SEM of three independent replicates. (F) 19-9-11 shcat-2 and scramble lines were cultured on Matrigel in mTeSR1 containing 12 μM CH. After 4 d, cells were immunostained for Nanog and Isl1. (Scale bar, 50 μm.)

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Staining, Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Temporal regulation of Wnt/β-catenin signaling promotes cardiac differentiation induced by serum or growth factors. (A) H9 cells on MEFs were treated with CH in hESC medium for 3 d before forming EBs. EBs were cultured in suspension using serum containing medium for 4 d before being transferred to 0.1% (wt/vol) gelatin-coated plates. The percentage of contracting EBs was determined visually. (B) H9 cells were cultured on Matrigel and treated with DMSO, 1 μM CH, or 1 μM BIO for 3 d before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1 in RPMI/B27-insulin medium using monolayer-directed differentiation. At day 15, the percentage of cTnT+ cells in culture was assessed by flow cytometry. #P < 0.005, CH versus DMSO or BIO versus DMSO; Student’s t test. (C) Schematic of the inducible shRNA construct for β-catenin knockdown and shRNA sequences targeting β-catenin. PH1TetO represents the human H1 promoter with Tet operator sequences. Red and green sequences are forward and reverse shRNA sequences of β-catenin, respectively; the loop sequence is shown in blue. (D) Representative phase-contrast and mCherry epifluorescence images of 19-9-11 cells transduced with lentiviral vectors containing the constructs described in C and selected by puromycin treatment. (E) 19-9-11 ishcat-1 and ishcat-2 cells were cultured in mTeSR1 containing 2 μg/mL dox. After 3 d, mRNA was collected, and β-catenin expression was evaluated by quantitative PCR. Error bars represent SEM of three samples. #P < 0.005, ishcat-1 versus iscramble or ishcat-2 versus iscramble; Student’s t test. (F) 19-9-11 ishcat-1 cells were cultured in mTeSR1 medium and were treated with BIO before exposure to 100 ng/mL activin A at day 0 and 5 ng/mL BMP4 at day 1, with 2 μg/mL dox added at the indicated times. Cells were analyzed for cTnT expression by flow cytometry 15 d after initiation of differentiation. Error bars represent SEM. of three independent experiments. #P < 0.005, for each time point versus no dox; Student’s t test.

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Cell Culture, Flow Cytometry, shRNA, Construct, Sequencing, Transduction, Expressing, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Modulating regulatory elements of Wnt signaling is sufficient for efficient and reproducible generation of human cardiomyocytes in the absence of growth factors. (A) Schematic of protocol for defined, growth factor-free differentiation of hPSCs expressing dox-inducible β-catenin shRNA to cardiomyocytes via treatment with small molecules. (B and C) 19-9-11 ishcat-1 cells were cultured as indicated in A with dox added 36 h after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry (B) or immunofluorescence (C). In B, the green histogram represents cTnT expression, and the red histogram is an isotype control. (Scale bar in C, 50 μm.) (D) 19-9-11 ishcat-2 cells were cultured as indicated in A, with dox added at different time points after treatment with 12 μM CH. At day 15, cells were analyzed for cTnT expression by flow cytometry. Error bars represent SEM of three independent experiments. *P < 0.05 and #P < 0.005, each time point versus no dox; Student’s t test. (E and F) 19-9-11 ishcat-1 cells were differentiated as described in A, with dox added 36 h after treatment with 12 μM CH. (E) At different time points, mRNA was collected, and RT-PCR analysis of pluripotent, mesendoderm, mesoderm, and cardiac gene expression was performed. (F) Day 7 cells were analyzed for Isl1 and Nkx2.5 expression by immunofluorescence. (Scale bar, 100 μm.)

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Expressing, shRNA, Cell Culture, Flow Cytometry, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling

doi: 10.1073/pnas.1200250109

Figure Lengend Snippet: Differentiation of hPSCs to cardiomyocytes via small-molecule modulation of regulatory elements of canonical Wnt signaling. GSK-3 inhibition stimulates mesoderm commitment of undifferentiated hPSCs. In the later stages of differentiation, expression of β-catenin shRNA or inhibition of Wnt ligand production stimulates cardiomyocyte differentiation.

Article Snippet: The pLKO.1-based β-catenin constitutive knockdown vectors shcat-1 and shcat-2 (plasmids 19761 and 19762; Addgene) and the β-catenin–inducible knockdown vectors ishcat-1 and ishcat-2 (Biosettia) were used for lentivirus particle production.

Techniques: Inhibition, Expressing, shRNA

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: TREM-1 expression in HK-2 cells treated with a TREM-1 vector or shRNA with or without LPS. HK-2 cells were transfected with a control vector, plasmid for TREM-1 expression, control siRNA or TREM-1-specific siRNA as described in the Materials and Methods section. Panel A: Representative PT-PCR results indicating suppression of TREM-1 by siRNA (TREM-1 siRNA) or overexpression of TREM-1 (TREM-1 DNA). Panels B & C: Representative immunoblotting results indicating suppression of TREM-1 by siRNA (TREM-1 siRNA) or overexpression of TREM-1 (TREM-1 DNA) from qualitative or quantitative analysis, respectively. * P < 0.05 compared with control cells without LPS; # P < 0.05 compared with control cells with LPS. The data presented are one representative assessment of triplicate assays with similar results.

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Expressing, Plasmid Preparation, shRNA, Transfection, Over Expression, Western Blot

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: The roles of TREM-1 in modulating HK-2 cell proliferation and apoptosis in response to LPS. HK-2 cells were transfected with a control vector, TREM-1 vector, control shRNA, or TREM-1-specific shRNA as described in the Materials and Methods section. The cells were then cultured for 72 h with or without LPS (10 µg/mL). Panel A: Quantitative comparison of cell proliferation in cells with or without LPS exposure. Panel B: Quantitative comparison of cell apoptosis in cells with or without LPS exposure. Panel C: Flow cytometry of cell apoptosis in cells with or without LPS exposure. * P < 0.05 compared with control cells without LPS; # P < 0.05 compared with control cells with LPS. The data presented are a representative assessment of triplicate assays with similar results.

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, shRNA, Cell Culture, Flow Cytometry

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: Inflammation and cytokine levels in supernatants of HK-2 cells treated with a TREM-1 vector or shRNA with or without LPS. The concentrations of sTREM-1 (panel A), IL-1β (panel B), TNFα (panel C), and IL-6 (panel D) were measured. HK-2 cells were transfected with a control vector, plasmid for TREM-1 expression, control siRNA or TREM-1-specific siRNA as described in the Materials and Methods section. * P < 0.05 compared with control cells without LPS; # P < 0.05 compared with control cells with LPS. The data presented are a representative assessment of triplicate assays with similar results.

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Plasmid Preparation, shRNA, Transfection, Expressing

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: LPS-induced HK-2 cell apoptosis was mediated by TREM-1. HK-2 cells were transfected with a plasmid containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA, followed by exposure to LPS (10 µg/mL). Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9 were assessed by immunoblotting as described in the Materials and Methods section. Panel A: Representative images showing the results of immunoblotting. Panel B: Average of three separate immunoblots for Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9. Vertical axis: ratio to control; horizontal axis: cells transfected with plasmids containing negative control vector/TREM-1 gene or negative control shRNA/TREM-1 shRNA. * P< 0.05 .

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Western Blot

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: LPS-induced HK-2 cell autophagy was mediated by TREM-1. HK-2 cells were transfected with a plasmid containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA, followed by exposure to LPS (10 µg/mL). Beclin-1, Atg-5, LC-3b, and p62 were assessed by immunoblotting as described in the Materials and Methods section. Panel A: Representative images of immunoblots. Panel B: Averages of three separate immunoblots for Beclin-1, Atg-5, LC-3b, and p62. Vertical axis: ratio to control; horizontal axis: cells transfected with plasmids containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA. * P< 0.05 .

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Western Blot

Journal: International Journal of Medical Sciences

Article Title: TREM-1 promoted apoptosis and inhibited autophagy in LPS-treated HK-2 cells through the NF-κB pathway

doi: 10.7150/ijms.50893

Figure Lengend Snippet: The NF-κB signaling pathway in LPS-treated HK-2 cells was regulated by TREM-1. HK-2 cells were transfected with plasmid containing a negative control vector/TREM-1 gene or negative control shRNA/TREM-1-specific shRNA, followed by exposure to LPS (10 µg/mL). P-p65, p65, P-IκBα, and IκBα were assessed by immunoblotting as described in the Materials and Methods section. Panel A: Representative images of immunoblots. Panel B: Average of three separate immunoblots for P-p65/p65 and P-IκBα/IκBα. Vertical axis: ratio to control; horizontal axis: cells transfected with plasmids containing negative control vector/TREM-1 gene or negative control shRNA/TREM-1 shRNA. * P< 0.05 .

Article Snippet: For TREM-1 depletion studies, TREM-1-specific siRNA (Santa Cruz, Cat# sc-42999), control (CON) shRNA plasmid (Santa Cruz, Cat# sc-108060), TREM-1 cDNA ORF clone (Sino Bio, Cat# HG10511-UT), or negative control vector (Sino Bio, Cat# CV011) was transfected into HK-2 cells using Lipofectamine 3000 (Thermo Fisher).

Techniques: Transfection, Plasmid Preparation, Negative Control, shRNA, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 is overexpressed in OSCCs patient samples and OSCC-derived cell lines. Total RNA from fresh samples and cell lines was converted to cDNA and subjected to qPCR. For gene expression analysis of tissue samples relative quantification was based on the comparison of a pool of five normal oral tissues, while the spontaneously immortalized but non-transformed epithelial cell line HGK was used as reference for comparison with OSCC-derived cell lines. The levels of HPSE1 mRNA were significantly higher in OSCC cell lines compared with HGK cells (A) . The high expression levels of protein HPSE1 were confirmed on OSCC-derived cell lines by Western Blot analysis (B) . The levels of HPSE1 mRNA were also significantly higher in OSCC tissue samples compared to normal oral mucosa (C) . Representative images in a high-power field (200×) of immunohistochemical analysis for HPSE1 in Normal oral tissue (D) and OSCC tissue preparations confirmed its higher expression at protein level; with a distinct cytoplasmic distribution and intensity in oral cancer samples expressing both higher (E) and lower (F) levels of HPSE1. Results were statistically determined by ANOVA followed by Tukey multiple comparison test, where ** p < 0.005, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Derivative Assay, Expressing, Transformation Assay, Western Blot, Immunohistochemical staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 overexpression discriminates OSCC and normal samples and predicts low patient survival. (A) Kaplan–Meier cumulative curves for disease-free survival of patients with OSCC as a function of HPSE1 expression, showing a 70% probability of death in patients with higher expression of HPSE1 compared with those with low expression. (B) Receiver operating characteristic (ROC) curves showing the ability of the HPSE1 overexpression distinguishes OSCC tumors from normal oral samples. (C) Kaplan–Meier of multivariate survival analysis combining HPSE1 expression levels with pT stages, where score 1: HPSE1 lower expressing samples associated with pT1/2, score 2: HPSE1 lower expressing samples associated with pT3/4 or higher HPSE1 with pT1/2, and score 3: HPSE1 higher expressing samples associated with pT3/4. These results confirmed that HPSE1 overexpression was significantly associated with higher grade tumors.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: HPSE1 knockdown and upregulation efficiency in OSCC cells. The efficiency of endogenous HPSE1 modulation was verified by Immunofluorescence (A) , Western Blot (B) and RT-qPCR (C) analyzes. For the loss-of-function strategy, SCC-9 cells were transduced with shRNA expressing a vector sequence against HPSE1 (shRNA HPSE1−) and empty vector (scrRNA control). For the gain-of-function strategy, SCC-9 cells were transduced with a ORF clone in a shuttle vector to enhance HPSE1 expression (orfRNA HPSE1+), along with mock-transduced cells as described in Methods . shRNA HPSE1− cells showed a significant reduction and orfRNA HPSE+ cells showed a significant upregulation of HPSE1 in both mRNA and protein levels compared to scrRNA control and parental mock-transduced SCC-9 control cells (pSCC9), without targeting sequences. Results were statistically determined by ANOVA, followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Immunofluorescence, Western Blot, Quantitative RT-PCR, Transduction, shRNA, Expressing, Plasmid Preparation, Sequencing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Downregulation of HPSE1 inhibits proliferation and enhances apoptosis of OSCC cells. Cells were subjected to MTT cell proliferation (A) , DNA content cell cycle analysis (B) , and apoptosis (C) assays. (A) Cell Proliferation Assay of the OSCC SCC9 cell line (Control) compared to empty vector SCC9 control clone (ScrRNA control), HPSE1 inhibitory/silencing SCC9-ShRNA HPSE1− clone, and the HPSE1 overexpressing SCC9-OrfRNA HPSE1+ clone. (B) Quantification of cell cycle analysis was performed by flow cytometry after staining with propidium iodide for the SCC-9 control cells and the respectives HPSE1-modulated clones. Abrogation of HPSE1 induced arrest of the SCC-9 cells cycle in G1 phase, and its upregulation significantly induced cell proliferation. (C) Flow cytometric analysis of apoptosis showed a remarkable increase in the number of apoptotic cells in HPSE1-silenced cells (ShRNA HPSE1−), while SCC-9 overexpressing HPSE1 (Orf-RNA HPSE1+) exhibited a reduction in apoptosis. (D) No significant differences in cell adhesion properties were observed among any transduced clones modulating HPSE1 expression, compared to parental mock-transduced cells (control). Plots compose experimental triplicate analysis and were statistically calculated using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: MTT Cell Proliferation, Cell Cycle Assay, Proliferation Assay, Plasmid Preparation, shRNA, Flow Cytometry, Staining, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 is associated with migration, invasion, ECM remodeling, and acquisition of EMT properties. (A) Photomicrographs of cell lines were taken 0, 24, and 48 h after wounding (40X). The average width of the lacunae was measured. Migration of SCC-9 cells was significantly decreased in HPSE1-silenced cells (ShRNA HPSE1−) and increased in HPSE1-upregulated clones (OrfRNA HPSE1+). Migration analysis based on this assay showed that cells with lower expression of HPSE1 closed the scratch wound significantly slower than the SCC9 Control cells. (B) Invasion of SCC-9 cells was significantly inhibited by HPSE1 knockdown, and significantly enhanced after its upregulation. (C) Analysing EMT markers, the downregulation of HPSE1 significantly induced the expression of E-Cadherin (E-CAD), while OrfRNA HPSE1+ SCC9 clones overexpressing heparanase had a significant increasing of Vimentin (VIM) and SNAIL expressions. (D) The upregulation of HPSE1 significantly enhanced the expressions of MMP2 and MMP9. All the graphs compile experimental triplicate analyses, and the results were obtained by ANOVA followed by Tukey assay, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Migration, shRNA, Clone Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Heparanase 1 Upregulation Promotes Tumor Progression and Is a Predictor of Low Survival for Oral Cancer

doi: 10.3389/fcell.2022.742213

Figure Lengend Snippet: Overexpression of HPSE1 enhances tumor neo-vascularization and induces VEGFA expression. HUVEC cells on a Miogel 2D layer co-culture. Photomicrograph of HUVEC endothelial cells added with preconditioned medium from OSCC cell lines and their respective clones after 12 h of experiment: (A) HUVEC cells incubated with preconditioned medium of SCC9-OrfRNA HPSE1+ clone overexpressing HPSE1 (100×); (B) HUVEC cells incubated with preconditioned medium of parental SCC9 (Control) (100×); (C) HUVEC cells incubated with preconditioned medium of SCC9-ShRNA HPSE1- clone with reduction of HPSE1 expression (100×). (D) Quantification of measures of the vessel circumference perimeter for each one of the conditions; the graph compiles two experimental triplicate analysis. (E) Gene expression analysis of endothelial growth factor VEGFA by qRT-PCR; the graph compiles folded expression values of relative quantification by ddCT obtained through comparison of clones OrfRNA HPSE1+ and ShRNA HPSE1− relative to the control SCC9 cells (normal reference = 1). Normalization of the analysis was performed using the endogenous control gene, PPIA. Statistical tests were performed using ANOVA followed by Tukey’s test, where * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Then, transduction of SCC-9 cells with control (scrRNA control cells) or shRNA HPSE1 sequences was performed using HuSH shRNA plasmid panel (short-hairpin RNA) following the manufacturer’s instructions (OriGene Technologies, United States/HPSE1, human, cat. No. TR307138).

Techniques: Over Expression, Expressing, Co-Culture Assay, Clone Assay, Incubation, shRNA, Quantitative RT-PCR

Journal: BMB Reports

Article Title: Adequate concentration of B cell leukemia/lymphoma 3 (Bcl3) is required for pluripotency and self-renewal of mouse embryonic stem cells via downregulation of Nanog transcription

doi: 10.5483/BMBRep.2018.51.2.219

Figure Lengend Snippet: Bcl3 is highly expressed in mouse embryonic stem cells. (A) Western blotting analysis of Bcl3 in mouse embryonic fibroblasts (MEF) and E14tg2a cells (E14). GAPDH was used as an internal control. (B) Western blot analysis of Bcl3 in E14 under the leukemia inhibitor factor (LIF) depletion conditions. After seeding, the cells were cultured for 3 days, and then were transferred in LIF depletion media. The samples were extracted at the indicated times. β-actin was used as an internal control. (C) Quantitative RT-PCR assay of Bcl3 in ESC-derived embryoid bodies. Embryoid bodies were formed by hanging drop culture methods and extracted at the indicated times. Data are normalized to β-actin and shown relative to E14 cells. ***P < 0.005 vs E14 cells. Error bars indicate the mean ± SEM (n = 3). P values were calculated by using one-way ANOVA. (D) Western blot analysis of Bcl3, Nanog, Sox2, and Oct4 in ESC-derived embryoid bodies. β-actin was used as an internal control.

Article Snippet: PVDF was probed with primary antibodies against Nanog (Bethyl), Bcl3, Sox2, Oct4, p53, GAPDH, β-actin (Santacruz) followed by the application of HRP-conjugated secondary antibodies.

Techniques: Western Blot, Control, Cell Culture, Quantitative RT-PCR, Derivative Assay

Journal: BMB Reports

Article Title: Adequate concentration of B cell leukemia/lymphoma 3 (Bcl3) is required for pluripotency and self-renewal of mouse embryonic stem cells via downregulation of Nanog transcription

doi: 10.5483/BMBRep.2018.51.2.219

Figure Lengend Snippet: Knockdown of Bcl3 attenuated pluripotency of mouse embryonic stem cells. (A) Western blot analysis of Bcl3 in E14 cells transfected with mock vector (shMock) and Bcl3 shRNA (shBcl3). GAPDH was used as an internal control. (B) Cell proliferation assay of shMock and shBcl3. The cells were seeded on a 24-well culture plate at a density of 3 × 10 3 cells/well and cultured for 3 days. ***P < 0.005 vs shMock. (C) Alkaline phosphatase staining of shMock and shBcl3. After AP staining, the colonies were scored, and the percentages of undifferentiated, mixed, and differentiated colonies were calculated. The bar graph shows the statistical evaluation. Error bars indicate the mean ± SEM (n = 3). P values were calculated by using two-way ANOVA. **P < 0.01 vs shMock. (D) Western blot analysis of pluripotent-related genes, Nanog, Oct4, and Sox2 in shMock and shBcl3. GAPDH was used as an internal control. (E) Quantitative RT-PCR of pluripotent related genes in shMock and shBcl3. The data are normalized to β-actin and expressed relative to shMock. The error bars indicate the mean ± SEM (n = 3). P values were calculated by using two-way ANOVA. **P < 0.01, ***P < 0.005 vs shMock.

Article Snippet: PVDF was probed with primary antibodies against Nanog (Bethyl), Bcl3, Sox2, Oct4, p53, GAPDH, β-actin (Santacruz) followed by the application of HRP-conjugated secondary antibodies.

Techniques: Knockdown, Western Blot, Transfection, Plasmid Preparation, shRNA, Control, Proliferation Assay, Cell Culture, Staining, Quantitative RT-PCR

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Identification of RNF168-associated proteins. A representative SDS–polyacrylamide gel electrophoresis of Flag-RNF168-associated proteins. Flag-tagged RNF168 was transfected in HEK293T cells and pull-down analysis was performed 48 h later. Protein bands were detected by silver staining. Protein bands were identified by mass spectrometry analysis following in-gel protease digestion. ( b ) HEK293T cells were transfected as indicated with HA-tagged RNF168 and Flag-TOP2α expression vectors. Cells were lysed and IP was performed using anti-Flag antibody. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) TOP2α, RNF168 and IgG (control) immunoprecipitates from HEK293T cells were examined by IB as indicated. ( b , c ) Data are representative of three independent experiments. ( d ) Cells treated with EdU were used for detection of localization patterns of TOP2α (Alexa Fluor 488) and RNF168 (Alexa Fluor 594) using confocal microscopy. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Polyacrylamide Gel Electrophoresis, Transfection, Silver Staining, Mass Spectrometry, Expressing, Control, Confocal Microscopy

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Representative agarose gel of in vitro kinetoplast DNA-based decatenation assays performed for 10 min with different amount of nuclear extracts from WT and Rnf168 −/− MEFs. Catenated and decatenated kDNA were separated by electrophoresis using 1% agarose gel. IB show Top2α's level in the total nuclear extracts used for this assay. ( b ) Representative data of the mitotic inhibition assay of decatenation G2 checkpoint in WT and Rnf168 −/− primary MEFs. Cells were treated with DMSO or ICRF-193 for 15 min and then incubated in culture media for an additional 2 h. The fraction of mitotic cells (pHH3 + ) was determined by flow cytometry. ( c ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of passage 1 primary MEFs (% pHH3 + cells post ICRF-193 treatment compared with DMSO-treated controls). * P <0.05. ( d ) Bar graphs represent the mean inhibition of mitotic index 2 h post ICRF-193 treatment of RIDDLE cells reconstituted with either HA-RNF168 or HA-empty vector as in c . * P <0.05. ( e ) Analysis of decatenation G2 checkpoint of Rnf168 −/− and WT 3T3 MEFs using the mitotic entry assay. Percentage of pHH3 + cells is shown at the indicated time post-treatment with colcemid in the presence of DMSO or ICRF-193. * P <0.05; Rnf168 −/− MEFs compared with WT MEFs 6 h post-ICRF-193 treatment. ( f ) Bar graphs represent the mean fraction of pHH3 + WT and Rnf168 −/− MEFs evading G2 arrest 6 h post-treatment with ICRF-193 compared with DMSO-treated cells as in e . * P <0.05. ( g ) Mitotic entry assay of decatenation G2 checkpoint in human RIDDLE cells reconstituted with HA-empty vector or HA-RNF168. Percentage of pHH3 + cells is shown at the indicated times post-treatment with colcemid with or without ICRF-193. * P <0.05, RIDDLE cells reconstituted with HA-empty vector compared with RIDDLE cells reconstituted with HA-RNF168 at 6 h post-ICRF-193 treatment. ( h ) Bar graphs represent the mean fraction of RIDDLE cells (reconstituted with HA-empty vector compared with those reconstituted with HA-RNF168) evading G2 arrest 6 h post ICRF-193 treatment compared with DMSO-treated controls as in f . * P <0.05. Three independent experiments in triplicates unless specified. Error bars in c , d , f and h represent mean±s.e.m.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Agarose Gel Electrophoresis, In Vitro, Electrophoresis, Inhibition, Incubation, Flow Cytometry, Plasmid Preparation

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a – d ) Sensitivity of WT and Rnf168 −/− MEFs to ICRF-193 ( a , b ) or etoposide ( c , d ) was determined using clonogenic assays. ( e , f ) Sensitivity of the human breast cancer cell lines T47D and MDA-MB-231 to etoposide was determined using clonogenic assays. ( a , c , e ) Data are presented as the mean±s.e.m. ( a , c , n >4; e , n =4). * P <0.05 for Rnf168 −/− MEFs compared with WT MEFs and T47D and MDA-MB-231 cells knocked down for RNF168 (sh.RNF168) compared with their respective controls (Sh.Ctr: ShRNA control). ( b , d ) Representative pictures of dishes showing surviving WT and Rnf168 −/− colonies post ICRF-193 ( b ) or etoposide ( d ) treatment. ( f ) Representative pictures of dishes showing surviving colonies of etoposide treated T47D and MDA-MB-231 cells.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: shRNA, Control

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector, and control HA-RNF168-reconstituted RIDDLE cells with TOP2α knock down were lysed and WCL subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies to detect ubiquitylated TOP2α. ( b ) Human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their control expressing ShRNA control (sh.Ctr) were examined for their level of ubiquitylated TOP2α as in a . ( c ) Rnf168 −/− , Brca1 −/− and WT MEFs were lysed and subjected to IP with anti-Top2α or IgG (control) antibodies. IPs from WCL were blotted with the indicated antibodies. ( d ) HEK293T cells were transfected with RNF168 (WT or mutant Rnf168-C21S), Flag-TOP2α and HA-Ub vectors as indicated. WCL were subjected to IP with anti-Flag, and IB analysis was performed using anti-HA antibody to detect ubiquitylated Flag-TOP2α. ( e ) Nuclear extracts from RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector were subjected to IP with anti-TOP2α or IgG (control) antibodies. Immunoprecipitates were blotted with the indicated antibodies against K63- and K48-Ub linkages. ( f ) In vitro ubiquitylation of recombinant TOP2α in the presence of recombinant RNF168 (500 ng for lane 5, 1 μg for lanes 2, 3 and 6 and 2 μg for lane7), UBE1 (E1), UBE2E2 (E2) and Ub. Nuc, nuclear extract; WCL, whole-cell lysate.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Plasmid Preparation, Control, Knockdown, Expressing, shRNA, Transfection, Mutagenesis, In Vitro, Recombinant

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) WT and Rnf168 −/− MEFs, and Top2α knockdown control MEFs were fixed and stained with DAPI. Representative cells with defective chromosome segregation, as indicated by chromosome bridges (arrow head) and micronuclei (arrow) are shown. ( b ) Histograms show quantification of cells with chromosome bridges or micronuclei (mean±s.e.m., n =3). * P <0.05. ( c ) Histograms show the fraction of RIDDLE cells reconstituted with HA-RNF168 or HA-empty vector that display micronuclei (mean±s.e.m., n =3). * P <0.05. ( d ; upper panels) Representative metaphase spreads showing undercondensed chromosomes in WT MEFs 24 h post ICRF-193 treatment (positive control), and in Rnf168 −/− DMSO-treated MEFs. ( d , lower panels) Representative metaphase spreads of DMSO-treated Rnf168 −/− MEFs complemented with RNF168-WT (WT) or the E3 ligase deficient RNF168-C21S (C21S). ( e ) Histograms show quantification of abnormal metaphase spreads with entangled or undercondensed chromosomes from the indicated cells (mean±s.e.m., n =3). * P <0.05 compared with WT MEFs. ( f ) Histograms present the mean fraction of MEFs evading ICRF-193-induced G2 arrest as compared with DMSO-treated controls (mean±s.e.m., n =3). Data are shown for WT MEFs, mock infected Rnf168 −/− MEFs and Rnf168 −/− MEFs complemented with RNF168-WT or RNF168-C21S. 3T3 MEFs were used for these experiments. Data shown are for 6 h post-treatment with colcemid±4 μM ICRF-193. * P <0.05 compared with WT MEFs. ( g ) Clonogenic assay was used to determine sensitivity to ICRF-193 of mock infected WT and Rnf168 −/− MEFs, as well as Rnf168 −/− MEFs complemented with RNF168 (WT or C21S mutant). Data are presented as the mean±s.e.m. ( n =4). * P <0.05 compared with WT MEFs. Scale bar, 20 μm.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Knockdown, Control, Staining, Plasmid Preparation, Positive Control, Infection, Clonogenic Assay, Mutagenesis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a ) Nuclear (Nuc) and chromatin (Chr) fractions prepared from WT, Rnf168 −/− and Brca1 −/− MEFs were analysed by IB for chromatin occupancy of Top2α. Additional IBs were performed with the indicated antibodies as controls. H4, histone H4. ( b ) Nuclear and chromatin fractions were prepared from the human breast cancer cell lines T47D and MDA-MB-231 knocked down for RNF168 (Sh.RNF168) and their controls (Sh.Ctr) and analysed by IB for the chromatin occupancy of TOP2α as in a . ( c ) HEK293T were transfected with Flag-TOP2α along with RNF168 (+) or empty vector (−) and their nuclear and chromatin fractions were prepared and examined by IB using the indicated antibodies. ( d ) A representative agarose gel showing decatenation activity of soluble nuclear and chromatin extracts from 2 WT and 2 Rnf168 −/− MEFs. In vitro kinetoplast DNA-based decatenation assay was performed for 20 min with different amounts of nuclear and chromatin extracts, and catenated and decatenated kDNA were separated by electrophoresis. IB using anti-Top2α was performed to show the level of Top2α present in each sample.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, In Vitro, Electrophoresis

Journal: Nature Communications

Article Title: RNF168 and USP10 regulate topoisomerase IIα function via opposing effects on its ubiquitylation

doi: 10.1038/ncomms12638

Figure Lengend Snippet: ( a , b ) HEK293T cells were transfected with Flag-RNF168 and HA-USP10 vectors ( a ) or Flag-TOP2α and HA-USP10 vectors ( b ) as indicated. Cells were lysed and IP was performed using anti-Flag and anti-HA antibodies. The resulting precipitates were subjected to IB analysis with the indicated antibodies. WCL, whole-cell lysate. ( c ) HEK293T cells were transfected with Flag-TOP2α, RNF168, HA-USP10 and Myc-Ub vectors, as indicated. IP using anti-Flag and WCL was subjected to anti-Ub IB analysis to detect TOP2α ubiquitylation. ( d ) WT MEFs with knockdown of Usp10 (Sh1 and Sh2) and WT controls were examined for the level of Top2α ubiquitylation. Top2α was immunoprecipitated from whole-cell extracts and examined by IB for its ubiquitylation level using anti-Ub. IP using IgG was used as a control. The indicated antibodies were used for IB. ( e ) HEK293T cells were transfected with Flag-TOP2α with or without RNF168, HA-Ub, USP10 and USP10-C424A as indicated. TOP2α chromatin occupancy in these cells was examined by IB using anti-Flag antibodies and chromatin fractions (Chr). IB analysis of the chromatin fractions is also shown for the indicated antibodies. ( f ) A simplified model of RNF168-mediated regulation of TOP2α ubiquitylation and decatenation function.

Article Snippet: After washing with 3% BSA in PBS, the sheep polyclonal against RNF168 (1:2,000; R&D systems cat# AF7217) and the rabbit polyclonal against TOP2α (Abcam) were detected using their respective secondary antibodies.

Techniques: Transfection, Knockdown, Immunoprecipitation, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: Hypoxia increased the production of S100 calcium-binding protein A8 (S100A8) in neuron and microglia and induced the release of S100A8 in SH-SY5Y cells. ( A , B ) S100A8 expression (red) were detected by immunocytochemical analysis in primary cultured neurons (NeuN, neuron marker) and cultured mixed glia (Iba1, microglial marker and GFAP, astrocyte marker) exposed to hypoxic conditions for 48 h. Scheme 25 μm. S100A8 expression was detected by western blot analysis in ( C , D ) SH-SY5Y cells and ( E , F ) BV-2 cells exposed to hypoxic conditions for 48 h. ( G , H ) S100A8 protein expression in BV-2 cells were confirmed by immunocytochemistry and ( I ) S100A8 release in SH-SY5Y was measured by enzyme-linked immunosorbent assay (ELISA) at 48 h after hypoxia. Values of * p < 0.05, ** p < 0.01, *** p < 0.001 versus control were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Binding Assay, Expressing, Cell Culture, Marker, Western Blot, Immunocytochemistry, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces pro-inflammatory cytokines and inflammation in BV-2 cells. BV-2 cells were stimulated with S100A8 (10 μg/mL) for 24 h. ( A ) The supernatant was collected and TNF-α and interleukin-6 (IL-6) analyzed by ELISA. ( B ) The protein and mRNA were extracted, and the expression levels of IL-1β were assessed by ELISA and RT-qPCR. ( C – E ) The protein was extracted, separated on 10% SDS-acrylamide gels (15 μg/lane) and transferred to nitrocellulose membrane. The protein expression level was detected by western blotting with anti-ERK1/2, anti-phospho-ERK1/2 (p-ERK1/2), anti-JNK and anti-p-JNK. ( F ) Cells were pre-treated with ERK inhibitor (PD98059, 20 μM), JNK inhibitor (SP600125, 10 μM) or the equivalent volume of DMSO for 1 h, then stimulated for 24 h with LPS or S100A8 for ELISA of TNF-α, IL-6. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, *** p < 0.001 versus control; ### p < 0.001 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Membrane, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 induces inflammasome priming by toll-like receptor (TLR)-4 receptors associated with ERK and JNK pathway in BV-2 cells. BV-2 cells were incubated for 24 h with LPS (1 μg/mL) or S100A8 (10 μg/mL) followed by Adenosine 5′-triphosphate disodium salt hydrate (ATP) (1 mM) for 1 h. ( A , B ) The NLRP3, ASC, and ( C , D ) cleaved caspase-1 were detected by western blotting. β-actin was used as an internal control. ( E , F ) BV-2 cells were lysed to whole lysates and IκB-α phosphorylation was analyzed by western blotting. ( G , H ) The translocation of nuclear factor- κB (NF-κB) was also detected by western blotting. BV-2 cells were lysed to cytosolic extracts and nucleic extracts. Lamin-B1 was used as internal controls. ( I , J ) BV-2 microglial cells were pre-treated with PD98059 (ERK inhibitor, 20 μM), SP600125 (JNK inhibitor, 10 μM), TAK-202 (TLR4 inhibitor, 10 μg/mL) or an equivalent volume of DMSO and stimulated for 24 h with LPS or S100A8. Cells harvested and lysed in RIPA buffer for western blotting of NLRP3. Results are from one experiment that is representative of at least three others. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ## p < 0.01 versus S100A8-treated sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Incubation, Western Blot, Control, Phospho-proteomics, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: S100A8 derived from neuronal cells induces NLRP3 inflammasome priming in microglia under hypoxic conditions. BV-2 cells were pre-treated with TAK-202 (TLR4 inhibitor, 10 μg/mL) for 1 h, then stimulated for 48 h in hypoxic condition with SH-SY5Y cells indirectly co-cultured in 0.4 μm pore transwell. ( A ) The protein expression level was detected by western blotting with NLRP3. β-actin was used as an internal control. ( B ) Quantitative analysis of NLRP3 levels. Data from three independent experiments are presented as the means ± S.D. Values of ** p < 0.01 versus control; # p < 0.05 versus co-cultured sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Derivative Assay, Cell Culture, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces apoptosis of neuronal cells in hypoxic condition. ( A , B ) SH-SY5Y cells incubated without or with S100A8 KD BV-2 cells for 48 h in hypoxic condition. Cleaved caspase-3 immunofluorescence images and were detected and quantitative analysis of the number of cleaved-caspase3-positive cells are shown in lower panel. ( C , D ) Representative Annexin-V/PI images were detected by flow cytometry. Quantitative analysis of the apoptotic rate of SH-SY5Y cells are shown in lower panel. ( E , F ) Primary neuron-glial mixed cells were transfected with S100A8 shRNA vector for 24 h followed by 48 h in hypoxic condition. Cells were harvested, and the expression protein levels of S100A8 and cleaved caspase-3 were analyzed by Western blotting. Data from three independent experiments are presented as the means ± S.D. Values of *** p < 0.001 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Incubation, Immunofluorescence, Flow Cytometry, Transfection, shRNA, Plasmid Preparation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Hypoxia-Induced S100A8 Expression Activates Microglial Inflammation and Promotes Neuronal Apoptosis

doi: 10.3390/ijms22031205

Figure Lengend Snippet: The expression of S100A8 in microglial cell induces the Cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE 2) pathway. BV-2 cells were transfected with S100A8 shRNA or Scramble vector. After 24 h, cells were incubated in hypoxic condition for 48 h. ( A ) The mRNA and ( B ) the protein levels of S100A8 and COX-2 were detected by real-time PCR and western blotting. ( C ) Secretion of PGE 2 level analyzed by ELISA. Data from three independent experiments are presented as the means ± S.D. Values of * p < 0.05, ** p < 0.01 versus control; # p < 0.05, ### p < 0.001 versus hypoxia-exposed sample were considered as statistically significant.

Article Snippet: S100A8, TNF-α, IL-6, IL-1β and PGE2 were quantitatively measured by an enzyme-linked immunosorbent assay (ELISA) using the human S100A8 Duoset ELISA kits, the mouse TNF-α, IL-6 and IL-1β DuoSet ELISA kits, and the PGE2 parameter assay kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Transfection, shRNA, Plasmid Preparation, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control

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

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: LAD2 mast cells were stably transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against MrgX2. (A) Western blotting was performed to determine MrgX2 expression in control and MrgX2 knockdown (KD) cells. (B) shRNA control and MrgX2 KD cells were stimulated with hBD2, hBD3, cortistatin (CST) or C3a and percent degranulation (β-hexosaminidase release) was determined. Data are mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Transduction, shRNA, Control, Western Blot, Expressing, Knockdown

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

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) RBL-2H3 cells stably expressing MrgX2 were stimulated with buffer, hBD2, hBD3 or cortistatin (CST) for 30 min and β-hexosaminidase release was measured. Data shown are representative of 3 similar experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01 and ** indicates p<0.001. RBL-2H3 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (B) hBD2, (C) hBD3 or (D) CST was determined. HEK293 cells stably expressing MrgX2 were loaded with Indo-1AM and Ca2+ mobilization in response to (E) hBD3 or (F) CST was determined. Traces shown are representative of 3 individual experiments.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Stable Transfection, Expressing

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

Article Title: ?-defensins activate human mast cells via Mas-related Gene-X2 (MrgX2)

doi: 10.4049/jimmunol.1300023

Figure Lengend Snippet: (A) BMMCs were transiently transfected with HA tagged MrgX2 (solid line) or control plasmid vector (broken line) and MrgX2 receptor expression level was analyzed using flow cytometry. A representative histogram is shown. (B) Control and MrgX2 expressing BMMCs were incubated with DNP specific mouse IgE (1 μg/mL, 16 h). Cells were exposed to buffer (control), CST, hBD3, mCRAMP or DNP-BSA (10 ng/mL) for 30 minutes and β-hexosaminidase release was measured. LAD2 cells were stimulated with mCRAMP and (C) intracellular Ca2+ mobilization or (D) degranulation was determined. Traces are representative of 3 independent experiments. Bar graphs represent mean ± SEM of three experiments. Statistical significance was determined by one-way ANOVA with Bonferroni's post test. * indicates p<0.01.

Article Snippet: MrgX2 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Incubation