Journal: PLoS ONE

Article Title: Studies on the non-invasive anticancer remedy of the triple combination of epigallocatechin gallate, pulsed electric field, and ultrasound

doi: 10.1371/journal.pone.0201920

Figure Lengend Snippet: The cell viability (24 h) was determined using MTT assay. (A) The HepG2 cells were triple treated with the EGCG (0 to 200 μM), 0.3 W/cm 2 US exposure (60 min), and consecutive 60 V/cm PEF for 24 h. The result revealed that the triple treatment could overcome the tolerance of cancer cells to EGCG and cause the death of HepG2 cells. (B) The triple treatment decreased the phosphorylation of Akt protein and increased the conversion of LC3-I to LC3-II in the HepG2 cells. The band intensities of LC3-II and LC3-I were quantified to calculate the LC3-II/LC3-I ratio. (C) The triple treatment triggered the activations of caspase-3 and PARP in the HepG2 cells. The cleaved PARP was quantified relative to the full-length PARP. β-actin was used as a loading control for each Western blot assay. (*** is used for P < 0.001).

Article Snippet: The human pancreatic cancer cell line PANC-1, the human hepatocellular carcinoma cell line HepG2, and the human embryonic kidney 293 (HEK293) were obtained from Bioresource Collection and Research Center (BCRC) (Hsinchu, Taiwan), and each STR-PCR profile for cell line authentication has been well recognized at BCRC.

Techniques: MTT Assay, Phospho-proteomics, Control, Western Blot

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: Primer sequences for qRT-PCR

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques:

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: The effect of the lentivirus-mediated shRNA interference of TNFR2 on TNF-α-stimulated EphB4 expression and osteogenic differentiation. a MC3T3-E1 cells stably transduced with lentiviral particles were selected with puromycin and named as pHBLV-TNFR2siRNA1 cells, pHBLV-TNFR2siRNA2 cells, pHBLV-TNFR2siRNA3 cells and pHBLV-NC cells, respectively. The mRNA levels of Tnfr2 were determined in these cells, among which the pHBLV-TNFR2siRNA1 cells displayed the highest TNFR2 gene silencing efficiency and were selected to continue the following studies. b TNFR2 protein levels in pHBLV-TNFR2siRNA1 cells and pHBLV-NC cells. c , d mRNA levels of Ephb4 , Runx2 and Bsp in pHBLV-TNFR2siRNA1 cells and pHBLV-NC cells cultured in the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α for 24 h ( c ) or 48 h ( d ). e , f Protein levels of EphB4, RUNX2 and BSP in pHBLV-TNFR2siRNA1 cells and pHBLV-NC cells cultured in the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α for 24 h ( e ) or 48 h ( f ). *, p < 0.05 vs. the pHBLV-NC group; **, p < 0.01 vs. the pHBLV-NC group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: shRNA, Expressing, Stable Transfection, Transduction, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: The effect of the impaired binding between TNF-α and TNFR2 on TNF-α-stimulated EphB4 expression and osteogenic differentiation. MC3T3-E1 cells were treated with an anti-mouse TNFR2/ CD120b/TNFRSF1B neutralizing antibody (TNFR2 NAb) at the concentration of 0.2 μg/ml, and were cultured in the osteogenic induction medium supplemented with or without 0.5 ng/ml TNF-α. Cells treated with 0.2 μg/ml of the normal rabbit IgG negative control antibody (control Ab) served as negative controls. (a) ALP activities were determined 7d or 14d after the treatment. (b, c) mRNA levels of Ephb4 , Runx2 and Bsp were determined after 24 h (b) or 48 h (c). (d, e) Protein levels of EphB4, RUNX2 and BSP were determined after 24 h (d) or 48 h (e). a, p < 0.05 vs. the control Ab group; b, p < 0.05 vs. the TNFR2 NAb group; c, p < 0.05 vs. the TNF-α + control Ab group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Binding Assay, Expressing, Concentration Assay, Cell Culture, Negative Control

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: The effect of inhibited EphB4 forward signaling on TNF-α-stimulated TNFR2 expression and osteogenic differentiation. (a) A potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, was used to suppress EphB4 forward signaling. MC3T3-E1 cells were pretreated with 200 nM NVP-BHG712 in the regular culture medium for 1 h. Cells were then incubated in osteogenic induction medium supplemented with 200 nM NVP-BHG712 and/or 0.5 ng/ml TNF-α for 7d or 14d. MC3T3-E1 cells cultured in osteogenic induction medium served as controls. The ALP activities were determined. (b, c) MC3T3-E1 cells were pretreated with 200 nM NVP-BHG712 for 1 h in the regular culture medium, and then incubated in osteogenic induction medium supplemented with 200 nM NVP-BHG712 and/or 0.5 ng/ml TNF-α. Cells cultured in osteogenic induction medium served as controls. mRNA levels of Tnfr2 , Runx2 and Bsp were determined after 24 h (b) or 48 h (c) of incubation. (d, e) MC3T3-E1 cells were pretreated with 200 nM NVP-BHG712 for 1 h in the regular culture medium, and then incubated in osteogenic induction medium supplemented with 200 nM NVP-BHG712 and/or 0.5 ng/ml TNF-α. Cells cultured in osteogenic induction medium served as controls. Protein levels of TNFR2, RUNX2 and BSP were determined after 24 h (d) or 48 h (e) of incubation. a, p < 0.05 vs. the control group; b, p < 0.05 vs. the NVP-BHG712 group; c, p < 0.05 vs. the TNF-α group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Expressing, Incubation, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: EphB4, TNFR2 and MAPK signaling pathways comprise a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation. a Levels of p38, p -p38, ERK1/2, p -ERK1/2, JNK1 + 2 + 3 and p -JNK1 + 2 + 3 in MC3T3-E1 cells treated with TNF-α for 0 min, 5 min, 15 min, 30 min and 60 min. b Levels of p38, p -p38, ERK1/2, p -ERK1/2, JNK1 + 2 + 3 and p -JNK1 + 2 + 3 in the pHBLV-TNFR2siRNA1 cells and the pHBLV-NC cells treated with or without 0.5 ng/ml TNF-α in regular culture medium for 15 min. c MC3T3-E1 cells were pretreated with or without 200 nM NVP-BHG712 in the regular culture medium for 1 h, and then 0.5 ng/ml TNF-α was added into the medium. The cells were incubated for another 15 min. Levels of ERK1/2 and p -ERK1/2 were determined. d-f MC3T3-E1 cells were cultured in the regular culture medium and pretreated with the ERK inhibitor U0126 (10 μM) for 1 h. The culture medium was then switched to the osteogenic induction medium supplemented with 0.5 ng/ml TNF-α and U0126 (10 μM). Cells treated without U0126 (10 μM) served as controls. ALP activities were determined 7d or 14d after the treatment ( d ). mRNA levels ( e ) and protein levels ( f ) of BSP and RUNX2 were determined 3 days after the treatment. *, p < 0.05 vs. the control group; **, p < 0.01 vs. the control group

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Incubation, Cell Culture

Journal: BMC Molecular and Cell Biology

Article Title: EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation

doi: 10.1186/s12860-020-00273-2

Figure Lengend Snippet: Schematic diagram of the EphB4, TNFR2 and ERK/MAPK signaling pathways. A low concentration of TNF-α first enhances the expression of EphB4, which in turn promoted the expression of TNFR2. The elevated TNFR2 level leads to the activation of the ERK signaling pathway, which eventually enhances the osteogenic differentiation of MC3T3-E1 cells. TNFR2, tumor necrosis factor receptor 2; TNF-α, tumor necrosis factor-alpha; MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase

Article Snippet: The anti-mouse primary antibodies used in this study were listed as following: RUNX2 (1:1000, catalog no. 12556S; CST, Danvers, MA, USA), BSP (1:1000, catalog no. 5468S CST, Danvers, MA,USA), EphB4 (1:1000, catalog no. A00690; Boster, China), TNFR2 (1:1000, catalog no. ab19139; abcam, Danvers, MA, USA), p38 (1:1000,catalog no. ab170099; abcam, Danvers, MA, USA), p -p38 (1:1000,catalog no. ab195049; abcam, Danvers, MA, USA), JNK1 + 2 + 3 (1:1000,catalog no. ab179461; abcam, Danvers, MA, USA), p -JNK1 + 2 + 3 (1:5000,catalog no. ab124956; abcam, Danvers, MA, USA), ERK1/2 (1:10000,catalog no. ab184699; abcam, Danvers, MA, USA), and p -ERK1/2 (1:8000,catalog no. ab76299; abcam, Danvers, MA, USA).

Techniques: Concentration Assay, Expressing, Activation Assay

Journal: Scientific Reports

Article Title: Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection

doi: 10.1038/srep45698

Figure Lengend Snippet: The mRNA levels in the cells were determined by real-time RT-PCR analysis. The ratios of target genes to GAPDH levels were determined. The data on HepG2 cells was normalized to 1. The data are presented as the mean ± S.D. (n = 4).

Article Snippet: HepG2 cells (a human hepatocellular carcinoma cell line, RCB1648, obtained from the JCRB Cell Bank), and those stably expressing NTCP (HepG2-hNTCP-C4) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection

doi: 10.1038/srep45698

Figure Lengend Snippet: ( a ) GFP expression levels following introduction of the GFP gene in iPS-HLCs. iPS-HLCs were transfected with pHMCA5-GFP using Lipofectamine2000 for 4 h or transduced with AdK7-CAGFP at 300 VP/cell for 2 h. GFP expression levels were evaluated 72-h after introduction of the GFP gene. ( b ) HBsAg and HBcrAg levels in the culture supernatants following introduction of the HBV genome. Human undifferentiated iPS cells, iPS-HLCs, and HepG2 cells were transduced with an Ad vector containing the HBV genome for 2 h. Following a total 72-h incubation, the levels of HBsAg and HBcrAg in the culture supernatants were determined by CLEIA. The data are presented as the mean ± S.D. (n = 3).

Article Snippet: HepG2 cells (a human hepatocellular carcinoma cell line, RCB1648, obtained from the JCRB Cell Bank), and those stably expressing NTCP (HepG2-hNTCP-C4) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Expressing, Transfection, Transduction, Plasmid Preparation, Incubation

Journal: Scientific Reports

Article Title: The citrus flavanone naringenin impairs dengue virus replication in human cells

doi: 10.1038/srep41864

Figure Lengend Snippet: Naringenin half maximal inhibitory concentration (IC 50 ) and selectivity index (SI) for anti-DENV activity in Huh7.5 cells.

Article Snippet: The human-derived Huh7.5 hepatoma cell line (ATCC PTA-8561) was grown in Dulbecco’s Modified Eagle Medium/nutrient mixture F-12 (D-MEM/F-12; Gibco, Grand Island, NY, USA) supplemented with 100 IU/μg/mL penicillin/streptomycin (Gibco, Grand Island, NY, USA) and 10% FBS at 37 °C in a humidified, 5% CO 2 controlled atmosphere.

Techniques: Concentration Assay, Activity Assay