Journal: Saudi Journal of Biological Sciences

Article Title: Live attenuated influenza vaccine induces broadly cross-reactive mucosal antibody responses to different influenza strains in tonsils

doi: 10.1016/j.sjbs.2023.103809

Figure Lengend Snippet: LAIV did not induce anti-H7N3 HA IgG antibody production. No significant level of specific anti-HA IgG antibody to H7N3 was detected compared with unstimulated cells (n = 15, p > 0.05). Means and standard errors are shown.

Article Snippet: Purified recombinant HA proteins of pH1N1 (A/California/04/2009,VR-1894DQ),sH1N1 (A/Brisbane/59/2007, VR-1742), sH3N2 (A/Brisbane/10/2007, VR-1881), aH5N1 (A/Vietnam/1203/2004, VR-1931), A/Hong Kong/8/68, VR-1679), and H7N3 (A/Canada/RV444/04, VR-1641) were from Biodefense and Emerging Infections Research Resources Repository (BEI resources ATCC (Manassas VA, USA).

Techniques:

Journal: iScience

Article Title: Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease.

doi: 10.1016/j.isci.2023.106686

Figure Lengend Snippet: Figure 1. Study design and Urinary extracellular vesicles (uEV) quality control (A) Study design for the next generation sequencing of uEV mRNAs to assess urine collections, quality and reproducibility (technical comparisons), origins of uEV transcripts (uEV on Tissue map), and discovery of candidate non-invasive markers for diabetic kidney disease (DKD marker candidates). (B–D) Transmission electron micrographs showing uEV of typical morphology and variable sizes; filamentous structures compatible with Tamm-Horsfall protein filaments are visible in the in-set B1. (E) Western blotting showing the presence of uEV-enriched markers CD9 and PDX in uEV preparations. (F) Representative electropherograms of RNA isolated from uEV and analyzed with bioanalyzer using Agilent Pico kit. An RNA peak between 25 and 200 nt was detected in all samples and with this characteristic shape passed quality control. (G) Examples showing signs of nucleic acid degradation (arrows) in uEV RNA samples and not passing quality control. 24 h (24h); albumin excretion rate (AER); blood pressure (BP); body-mass index (BMI); estimated glomerular filtration rate (eGFR); waist-to-hip ratio (WHR); Genotype-Tissue Expression (GTEx) project; glycated hemoglobin (HbA1C); Lymph Node Carcinoma of the prostate (LnCaP), macroalbuminuria (Macro); microalbuminuria (Micro); non-diabetic control (Control); normoalbuminuria (Normo); messenger RNA sequencing (mRNAseq); Podocalyxin (PDX), type 1 or type 2 diabetes (T1D, T2D); urinary extracellular vesicles (uEV); principal component analysis (PCA); single-nucleus RNA sequencing (snRNA-seq).

Article Snippet: DIREVA samples were collected with PI and centrifuged at 1800g for 10 min at +4 C before freezing as above, and FinnDiane samples were collected either with or completely without these steps. iBEAt samples all contained PI (Complete ULTRA, Mini, EDTA-free, Roche; half tablet per 50 ml urine) and were centrifuged at 3000g for 10 min at RT, after which citrate-EDTA buffer was added (1,0 M citrate, 0,1 M EDTA; 2,5 ml per 45 ml urine) before freezing at -80 C. The uEV were isolated from 30 ml of urine (DIREVA and part of FinnDiane) as above except using a SW-32 rotor (129 168 gmax, k-factor 276, Beckmann-Coulter) and from 8 ml of urine (iBEAt and part of FinnDiane) as described (Barreiro et al.23) using a 70.1 ti rotor (82 656 gmax, k-factor 202, Beckman Coulter, Inc., Brea, CA) for 2 h 30 min at 30,000 rpm at +4 C and no wash. uEV protein and particle quality control Urinary EV samples were analyzed as explained previously.22,66 Briefly, Western blotting of uEVmarker proteins was performed using antibodies against CD9 (SC-13118, Santa Cruz) and podocalyxin (clone 3D3, Novus Biologicals).

Techniques: Control, Next-Generation Sequencing, Marker, Transmission Assay, Western Blot, Isolation, Expressing, RNA Sequencing

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a , b The expression of glycolysis-related genes was detected by RT-qPCR in both B7-H3-overexpressing HCT116 ( a ) and RKO ( b ) cells. c , d The mRNA level of HK2 was detected by RT-qPCR in both HCT116 ( c ) and RKO ( d ) cells after transfection with siRNA NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. e HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both HCT116 and RKO cells after transfection with NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. β-actin served as a loading control. f HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. β-actin served as a loading control. g Schematic representation of the proposed B7-H3/STAT3/HK2 axis. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Western Blot, Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a The protein level of HK2 in B7-H3-overexpressing HCT116 or RKO cells after transfection with siRNA negative control (NC) or HK2 siRNA transfection were analyzed by western blot. β-actin served as a loading control. b , c Glucose consumption ( b ) and lactate production ( c ) were measured in B7-H3-overexpressing HCT116 or RKO cells transfected with NC or HK2 siRNA. d , e Glucose consumption ( d ) and lactate production ( e ) were measured in B7-H3-overexpressing HCT116 or RKO cells treated with PBS or 2-DG. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Transfection, Negative Control, Western Blot, Control

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a , b Colony formation assay of B7-H3-overexpressing HCT116 or RKO cells exposed to L-OHP. The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. c , d Cell viability was analyzed using the CCK-8 kit. B7-H3-overexpressing HCT116 or RKO cells showed resistance to L-OHP ( c ) or 5-FU ( d ). The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. e B7-H3-overexpressing HCT116 or RKO cells showed less cell apoptosis than control cells (EV) after L-OHP treatment. The effect of B7-H3 on cell apoptosis was abolished by HK2 siRNA or 2-DG. f The protein levels of Bcl-2 and Bax were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. The effect of B7-H3 on Bcl-2 and Bax expression was abolished by HK2 siRNA. β-actin served as a loading control. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Colony Assay, CCK-8 Assay, Control, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: B7-H3 promotes aerobic glycolysis and chemoresistance in colorectal cancer cells by regulating HK2

doi: 10.1038/s41419-019-1549-6

Figure Lengend Snippet: a Images of IHC analysis of B7-H3 and HK2 protein expression and hematoxylin and eosin (H&E) staining of CRC ( n = 126) tissue sections. One representative image is shown. b , c B7-H3 ( b ) and HK2 ( c ) protein expression based on their staining index in nonmalignant adjacent tissues (NAT) and CRC specimens. d Correlation analysis of the staining index of expression levels of B7-H3 and HK2 protein in human CRC specimens ( n = 126). e , f B7-H3 ( e ) and HK2 ( f ) protein expression based on their staining index in CRC specimens at different clinical stages. Values are expressed as means (SEMs). * P < 0.05

Article Snippet: The antibodies for the western blot analysis in this study were as follows: goat anti-human 4IgB7-H3 (R&D Systems, #AF1027), mouse anti-human HK2 (Novus Biologicals, #NBP1-51643), rabbit anti-human/mouse STAT3 (CST, #12640, MA, USA), rabbit anti-human/mouse Phospho-STAT3 (pSTAT3) (CST, #9145), rabbit anti-human Bcl-2 (abcam, #ab32124), rabbit anti-human/mouse Bax (abcam, #ab32503) and mouse anti-human/mouse β-actin (CST, #3700).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint

doi: 10.1371/journal.pone.0131047

Figure Lengend Snippet: (A) Analysis of RhoH protein expression by western blot in RhoH -/- , RhoH -/- RhoH Tg , and RhoH +/+ thymocytes. (B, C) Analysis of RhoH -/- , RhoH -/- RhoH Tg , and RhoH +/+ thymocytes by flow cytometry. Two parameter plots show CD4 versus CD8 surface staining of thymocytes (upper), and CD25 versus CD44 surface staining on CD4 - CD8 - (DN) cells (lower). Numbers indicate percentage of cells in the selected area. Bar graphs represent average cell number and frequency of indicated thymocyte subsets calculated from six mice per group. (D) Single parameter histogram plots show CD2 and CD5 staining in DP thymocytes gated on CD4 + CD8 + cells (n = 6). Data are shown as mean +SD of more than four mice representative of independent experiments. *P<0.05, **P<0.01, ***P<0.001

Article Snippet: RhoH mouse monoclonal antibody (3D3) was purchased from Novus Biologicals (Littleton CO).

Techniques: Expressing, Western Blot, Flow Cytometry, Staining

Journal: PLoS ONE

Article Title: Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint

doi: 10.1371/journal.pone.0131047

Figure Lengend Snippet: Analysis by flow cytometry of thymocytes and splenocytes from RhoH +/+ RhoH Tg (red) and RhoH +/+ (blue) mice. Representative two parameter plots show CD4 versus CD8 staining on thymocytes (A, n = 8) and splenocytes (E, n = 13). (B) Representative single-parameter histogram plots show intracellular staining of Phospho-src (pY416) gated on CD4 - CD8 - CD44 - CD25 + (DN3) cells (n = 5). Representative single-parameter histogram plots show cell surface staining of CD2 and CD5 antigens on DP cells from RhoH +/+ RhoH Tg and RhoH +/+ mice in either MHC +/+ (C, n = 6) or MHC -/- (D, n = 5) background. Solid line and dashed line represent RhoH +/+ and RhoH +/+ RhoH Tg , respectively. (F, G) Flow cytometric analysis of CD44 versus CD62L expression profile on splenic CD4 + T (F) or CD8 + T (G) cells gated on TCRβ + T cells (n = 10). Data are shown as mean +SD and samples were from more than four independent experiments. **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: RhoH mouse monoclonal antibody (3D3) was purchased from Novus Biologicals (Littleton CO).

Techniques: Flow Cytometry, Staining, Expressing

Journal: PLoS ONE

Article Title: Overexpression of RhoH Permits to Bypass the Pre-TCR Checkpoint

doi: 10.1371/journal.pone.0131047

Figure Lengend Snippet: (A) FACS analysis of the in vitro differentiation of thymocytes to the DP stage. DN3 cells from Rag2 -/- , Rag2 -/- RhoH tg , and Rag2 +/+ C57B6 mice were differentiated for 14 days with the stromal cell line TSt-4/Dll-1 in the presence of IL-7. Representative two parameter plots show CD4 versus CD8 staining on cultured thymocytes at the indicated days. Results shown are from one out of three independent experiments. (B) DN3 cells were cultured with vehicle alone or a pharmacological inhibitor of Lck at the indicated concentration. Bar graphs represent the percentages of DP thymocytes compared with the vehicle control group. Data are representative of more than three independent experiments. ***P<0.001.

Article Snippet: RhoH mouse monoclonal antibody (3D3) was purchased from Novus Biologicals (Littleton CO).

Techniques: In Vitro, Staining, Cell Culture, Concentration Assay, Control

Journal: iScience

Article Title: Genome-wide mRNA profiling in urinary extracellular vesicles reveals stress gene signature for diabetic kidney disease.

doi: 10.1016/j.isci.2023.106686

Figure Lengend Snippet: Figure 1. Study design and Urinary extracellular vesicles (uEV) quality control (A) Study design for the next generation sequencing of uEV mRNAs to assess urine collections, quality and reproducibility (technical comparisons), origins of uEV transcripts (uEV on Tissue map), and discovery of candidate non-invasive markers for diabetic kidney disease (DKD marker candidates). (B–D) Transmission electron micrographs showing uEV of typical morphology and variable sizes; filamentous structures compatible with Tamm-Horsfall protein filaments are visible in the in-set B1. (E) Western blotting showing the presence of uEV-enriched markers CD9 and PDX in uEV preparations. (F) Representative electropherograms of RNA isolated from uEV and analyzed with bioanalyzer using Agilent Pico kit. An RNA peak between 25 and 200 nt was detected in all samples and with this characteristic shape passed quality control. (G) Examples showing signs of nucleic acid degradation (arrows) in uEV RNA samples and not passing quality control. 24 h (24h); albumin excretion rate (AER); blood pressure (BP); body-mass index (BMI); estimated glomerular filtration rate (eGFR); waist-to-hip ratio (WHR); Genotype-Tissue Expression (GTEx) project; glycated hemoglobin (HbA1C); Lymph Node Carcinoma of the prostate (LnCaP), macroalbuminuria (Macro); microalbuminuria (Micro); non-diabetic control (Control); normoalbuminuria (Normo); messenger RNA sequencing (mRNAseq); Podocalyxin (PDX), type 1 or type 2 diabetes (T1D, T2D); urinary extracellular vesicles (uEV); principal component analysis (PCA); single-nucleus RNA sequencing (snRNA-seq).

Article Snippet: Antibodies Mouse monoclonal against CD9 Santa Cruz Cat# SC-13118; RRID: AB_627213 Mouse monoclonal against Podocalyxin (clone 3D3) Novus Biologicals Cat# NBP2-25219; RRID: N/A

Techniques: Control, Next-Generation Sequencing, Marker, Transmission Assay, Western Blot, Isolation, Expressing, RNA Sequencing

Journal: Oncotarget

Article Title: Downregulation of the phosphatase JKAP/DUSP22 in T cells as a potential new biomarker of systemic lupus erythematosus nephritis

doi: 10.18632/oncotarget.11419

Figure Lengend Snippet: A. ELISAs of serum IFN-γ, IL-17, IL-6, and TNF-α levels in 12- and 24-week-old Lck-JKAP-C88S transgenic mice and age-matched wild-type mice. B. ELISAs of serum anti-nuclear antibody (ANA) and anti-dsDNA levels in 24-week-old Lck-JKAP-C88S transgenic mice and age-matched wild-type mice. The ANA and anti-dsDNA levels are presented relative to the value from one of control wild-type mice. C. Photomicrographs of the spleen and kidney tissues from 24-week-old Lck-JKAP-C88S transgenic mice and age-matched wild-type mice stained with hematoxylin-eosin. Scale bar: 100 μm. D. Representative micrographs of periodic acid-Schiff (PAS)-stained kidney sections from 52-week-old Lck-JKAP-C88S transgenic mice and their wild-type littermates. Arrows, brush borders. Arrowheads, protein droplets. At least four mice were analyzed for each group. Two-tailed Student's t -test, *, P < 0.05. Bars show the mean ± SEM. WT, wild-type; C88S, Lck-JKAP-C88S transgenic mice.

Article Snippet: Anti-JKAP antibody (clone #3D3) purchased from Abnova, anti-phospho-Y394-Lck antibody (#2101) purchased from Cell Signaling, anti-myc antibody (#9E10) purchased from Millipore, and anti-β-actin antibody (#AC-74) purchased from Sigma-Aldrich were used for immunoblotting.

Techniques: Transgenic Assay, Control, Staining, Two Tailed Test

Journal: Oncotarget

Article Title: Downregulation of the phosphatase JKAP/DUSP22 in T cells as a potential new biomarker of systemic lupus erythematosus nephritis

doi: 10.18632/oncotarget.11419

Figure Lengend Snippet: A. Kidney tissue fluids from 6-month-old wild-type and Lck-JKAP-C88S transgenic mice were subjected to the cytokine array analyses. The cytokines whose levels were increased in Lck-JKAP-C88S transgenic mice are labeled by colored rectangles. WT, wild-type; C88S, Lck-JKAP-C88S transgenic mice. B. Primary splenic T cells purified from 12-week-old wild-type mice and JKAP knockout (KO) mice were cultured for 3 days without any stimulation. The C5a, IL-16, soluble ICAM-1, and soluble VCAM-1 levels in the supernatants were determined by ELISA assays. Two-tailed Student's t -test, *, P < 0.05. Bars show the mean ± SEM.

Article Snippet: Anti-JKAP antibody (clone #3D3) purchased from Abnova, anti-phospho-Y394-Lck antibody (#2101) purchased from Cell Signaling, anti-myc antibody (#9E10) purchased from Millipore, and anti-β-actin antibody (#AC-74) purchased from Sigma-Aldrich were used for immunoblotting.

Techniques: Transgenic Assay, Labeling, Purification, Knock-Out, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test