Journal: PLoS ONE

Article Title: α-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways

doi: 10.1371/journal.pone.0044093

Figure Lengend Snippet: ( A ) HL60 and ( B ) K562 cells were treated with and without α-tomatine (5 µM) for 12 hr, 18 hr, 24 hr, and 30 hr. Cells were then fractionated into nuclear components, and the protein expressions of AIF and nucleolin (nuclear loading control) were evaluated by Western blot analysis. ( C ) HL60 and ( D ) K562 cells were treated with α-tomatine at the indicated concentrations and time. Survivin and actin protein levels were detected by Western blot analysis. Data are expressed from at least three separate determinations.

Article Snippet: The slides were incubated with the diluted primary antibody, survivin or AIF (Cell Signaling, Beverly, MA) at 4°C overnight.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: α-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways

doi: 10.1371/journal.pone.0044093

Figure Lengend Snippet: HL-60 cells were ectopically implanted into SCID mice and when the tumor size reached 100 mm 3 , the mice were injected with 5 mg/kg (q2d, i.p.) doses of α-tomatine. ( A ) Effects of α-tomatine on tumor volume and the body weights of mice were studied. The growth curves are the means of the tumor sizes measured for each group (n = 5). ( B ) The tumors were then excised and processed for immunohistochemical staining. The upper lanes (a.c.e.g and i) are the control, and the down lanes (b.d.f.h and j) are the treated group, with α-tomatine (5 mg/kg). a,b: Hematoxylin and eosin staining; c,d,e and f staining for AIF (brown) ; g,h,i and j staining for survivin (brown). c,d,g and h are under 200× magnification; a,b,e,f and j are under 1000× magnification. ( C ) Western blot analysis was performed for AIF and survivin expressions together with actin as a loading control from randomly selected tumor in each of the control and 5 mg/kg α-tomatine treatment groups.

Article Snippet: The slides were incubated with the diluted primary antibody, survivin or AIF (Cell Signaling, Beverly, MA) at 4°C overnight.

Techniques: Injection, Immunohistochemical staining, Staining, Western Blot

Journal: Neuro-Oncology Advances

Article Title: Novel genetically engineered H3.3G34R model reveals cooperation with ATRX loss in upregulation of Hoxa cluster genes and promotion of neuronal lineage

doi: 10.1093/noajnl/vdad003

Figure Lengend Snippet: ATRX loss significantly increases tumor latency in the absence of H3.3G34R overexpression. (A) Tumor grades for all injection groups. (B) Kaplan–Meier survival curves for indicated injection groups. (C) Representative GFAP and H&E staining of ependymal differentiation in H3.3G34R-GFP overexpressing samples. (D) Ependymal differentiation incidence for H3.3G34R ATRX WT vs H3.3G34R ATRX KO (* P < .05, Fisher’s exact test). (E) Representative EMA staining of H3.3G34R-GFP overexpressing samples. (F) Representative Iba1 staining of ATRX WT samples.

Article Snippet: IHC was performed using a Vectastain Elite kit (Vector Laboratories #AK-5001) as described previously with the following antibodies: anti-PDGFRA (Cell Signaling Technology, #3174T, 1:1000), anti-pERK1/2 (ABclonal, #AP0472, 1:100), anti-Iba1/AIF-1 (Cell Signaling Technology, #17198T, 1:1500), and anti-H3.3G34R (abcam, #ab254402, 1:1000).

Techniques: Over Expression, Injection, Staining

Journal: PLOS Pathogens

Article Title: Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response

doi: 10.1371/journal.ppat.1011170

Figure Lengend Snippet: A) THP-1 cells were infected with the MeV expressing the One-STrEP-tagged MeV-V protein or the One-STrEP-tagged CH protein used as control. Expression of One-STrEP-tagged MeV-V proteins were determined by Western blot in total cell lysates (left panel) or following affinity purification on Strep-Tactin Sepharose beads using a rabbit polyclonal anti-V rAb kindly provided by Dr. Kaoru Takeuchi (right panel). ALAS1 protein was detected only with MeV expression of One-STrEP-tagged MeV-V proteins prior chromatography purification. B) Purity control of cellular fractions by immunoblotting, using β-actin to indicate the cytosol, AIF (apoptosis inducing factor) for mitochondria and MEK1/2 as a marker of cytosolic proteins. kDa: kilo Dalton; p: precursor, m: mature. Arrows represent the two forms of ALAS1. C) Quantification of ALAS1 protein upon MeV or MeV-ΔV infections at MOI 1 and 3. Mean values and s.e.m. were calculated for five independent experiments (unpaired two-side Student’s t-test, ***, p < 0.005). D) Quantification of ALAS1 protein upon transfection with 0, 0.5 and 1μg of MeV-V plasmid. NT: non transfected, jP: jetPRIME. Mean values and s.e.m. were calculated for two independent experiments. Unpaired two-side Student’s t-test, **, p < 0.05, ***, p < 0.005.

Article Snippet: Anti-rabbit MEK1/2 (#8727), anti-rabbit AIF (#5318) and anti-rabbit Histone H3 (#4499) were from cell signaling, anti-rabbit ALAS1 (#MA5-35584) were from Invitrogen.

Techniques: Infection, Expressing, Western Blot, Affinity Purification, Chromatography, Purification, Marker, Transfection, Plasmid Preparation

Journal: BMC Cell Biology

Article Title: A potential role of the JNK pathway in hyperoxia-induced cell death, myofibroblast transdifferentiation and TGF-β1-mediated injury in the developing murine lung

doi: 10.1186/1471-2121-12-54

Figure Lengend Snippet: Effect of JNK inhibition on fetal alveolar interstitial fibroblasts (AIF) in hyperoxia . AIF were exposed to 21% up to 95% O2 over 24 h and 48 h, and cell proliferation was assessed by thymidine uptake ( 2A ). Total and P-JNK were assessed ( 2B ) and P-JNK staining was confirmed by immunocytochemistry ( 2C ). Use of JNKi (CEP-1347) blocked the hyperoxia-induced decrease in lipid droplet staining (red fluorescence) and an increase in α-SMA staining (green fluorescence), two key markers of AIF-to-MYF transdifferentiation ( 2D ) and diminished P-JNK ( 2E ). Use of JNKi (SP600125) diminished P-JNK ( 2F ) and blocked the hyperoxia-induced decrease in peroxisome proliferator-activated receptor γ (PPARγ) and adipocyte differentiation-related protein (ADRP) levels ( 2G ), and the accompanying increase in fibronectin and LEF-1 levels ( 2H ). O2: oxygen; JNKi: JNK inhibitor (SP600125 for A549 and AIFs, CEP-1347 for AIFs). All values represent a minimum of 4 measurements in each group and all figures are illustrative of a minimum of 4 experiments. * P < 0.05, ** P ≤ 0.02, # P ≤ 0.01.

Article Snippet: For the AIFs, the antibodies utilized were JNK and phospho-JNK (catalog # 9252 and 9251, respectively, Cell Signaling, Danvers, MA), PPARγ (catalog # sc-7196, Santa Cruz, CA), ADRP [a gift from (late) Dr. Constantine Londos, NIDDK], fibronectin (catalog # sc-8422, Santa Cruz, CA), LEF-1 (catalog #, sc-28687, Santa Cruz, CA).

Techniques: Inhibition, Staining, Immunocytochemistry, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: DL0410 Alleviates Memory Impairment in D-Galactose-Induced Aging Rats by Suppressing Neuroinflammation via the TLR4/MyD88/NF- κ B Pathway

doi: 10.1155/2021/6521146

Figure Lengend Snippet: Effect of DL0410 on microglial activation and astrocyte activation stained by IHC and analyzed by Western blot with Iba-1 and GFAP. Representative staining images of Iba-1 IHC staining in the hippocampus (a) and cortex (b) of rats. Representative staining images of GFAP IHC staining in the hippocampus (c) and cortex (d) of rats. The Western blot detection of Iba-1 expression and the quantitative analysis in the hippocampus (e) and cortex (f) of rats. The Western blot detection of GFAP expression and the quantitative analysis in the hippocampus (g) and cortex (h) of rats. Bar = 100 μ m. Values are mean ± SEM ( n = 6). # p < 0.05, ## p < 0.01, ### p < 0.001 vs. the control group, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the model group.

Article Snippet: The radio immune precipitation assay (RIPA) buffer, primary antibodies against β -actin (#3700), inducible nitric oxide synthase (iNOS) (#13120), cyclo-oxygenase-2 (COX2) (#12282), MyD88 (#4283), TRAF6 (#8028), NF- κ B p65 (#8242), phosphorylated- (p-) NF- κ B p65 (#3033), Histone H3 (#4499), I κ B α (#4814), p-I κ B α (#2859), ionized calcium-binding adapter molecule 1 (Iba-1) (#17198), occludin (#91131), claudin-1 (#13255), neuronal nuclear antigen (NeuN) (#24307), SOD1 (#37385), and SOD2 (#13141) were the products of Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Staining, Western Blot, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Coxiella burnetii Induces Apoptosis during Early Stage Infection via a Caspase-Independent Pathway in Human Monocytic THP-1 Cells

doi: 10.1371/journal.pone.0030841

Figure Lengend Snippet: Cells were infected with NMII at MOI of 50 for 24 h. Infected cells were then washed and incubation was continued for 48 h. Infected and uninfected THP-1 cells were fixed with paraformaldehyde and permeabilized. Intracellular C. burnetii were stained by IFA with rabbit anti- Coxiella polyclonal antibodies and viewed using a fluorescence microscope. Panel 1A, IFA staining of NMII infected THP-1 cells. Upper panel: uninfected control cells; lower panel: NMII infected cells. 1 Hoechst staining for host cell DNA; 2 Cells stained with anti- Coxiella antibodies; and 3 Merge. Panel 1B, growth rate of NMII in THP-1 cells. NMII infected and uninfected monocytic THP-1 cells were directly lysed at 24, 48 or 72 h post infection. DNA was extracted and used as a template to quantify the number of C. burnetii com1 gene copies by real-time PCR.

Article Snippet: After blocking in 5% non-fat dried milk at room temperature for 1 hour, membranes were probed with rabbit polyclonal anti-AIF antibody (Cell Signaling Technology Inc., MA).

Techniques: Infection, Incubation, Staining, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Coxiella burnetii Induces Apoptosis during Early Stage Infection via a Caspase-Independent Pathway in Human Monocytic THP-1 Cells

doi: 10.1371/journal.pone.0030841

Figure Lengend Snippet: Approximately 1×10 6 NMII infected or uninfected cells were double stained with Annexin-V-FITC and PI. Panel 3A, representative FACS scatter plots of THP-1 cells. Fluorescence was detected using a fluorescence-activated cell sorter to analyze necrotic (PI+), non-apoptotic (negative for both dyes), early apoptotic (Annexin+/PI−), and late apoptotic cells (Annexin+/PI+). Panel 3B, percentages of apoptotic cells in NMII infected THP-1 cells. Data shown represents the Mean±SE from at least three independent experiments. * denotes significant differences (*** p <0.001) between infected and uninfected cells at each time point post infection. Panel 3C, double staining of intracellular C. burnetii and apoptotic cell DNA. Intracellular C. burnetii was stained by IFA with rabbit anti- Coxiella polyclonal antibodies and apoptotic host nuclei were stained with TUNEL staining. Upper panel: Cells stained with anti- Coxiella antibodies; Middle panel: TUNEL stating of apoptotic host nuclei; Lower panel: Merge. From left to right 1) Normal cell control; 2) Staurosporine (1 µm) treated apoptotic control cell; 3) NMII infected cells at 24 h post infection; 4) NMII infected cells at 48 h post infection; 5) NMII infected cells at 72 h post infection.

Article Snippet: After blocking in 5% non-fat dried milk at room temperature for 1 hour, membranes were probed with rabbit polyclonal anti-AIF antibody (Cell Signaling Technology Inc., MA).

Techniques: Infection, Staining, Fluorescence, Double Staining, TUNEL Assay

Journal: Cancer Management and Research

Article Title: Suppression of miR-21-3p enhances TRAIL-mediated apoptosis in liver cancer stem cells by suppressing the PI3K/Akt/Bad cascade via regulating PTEN

doi: 10.2147/CMAR.S183328

Figure Lengend Snippet: Antibodies used for the study

Article Snippet: 13 , AIF antibody , Cell Signaling Tech.

Techniques: