Journal: PLoS ONE

Article Title: The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0056399

Figure Lengend Snippet: Representative photographs of monolayer differentiated cells (Days 2 and 7: original magnification, x200) (A). Representative electrophoretic mobility shift assays (EMSA) shows NF-κB binding activity in the undifferentiated iPS cells (FGF2+ and feeder+) and monolayer differentiated cells (FGF2- and feeder-). Proximal tubular epithelial cells (PTECs) were used as control cells (differentiated cells) for the EMSA. Results of 2 independent experiments are shown. In the last lane, the competition assays were performed using the undifferentiated iPS cells (B). Western blot analysis of actin, Oct3/4, NANOG, WT-1, and Pax-2 in the undifferentiated iPS cells (FGF2+ and feeder+) and monolayer differentiated cells (FGF2- and feeder-). Feeder cells (SNL) were used as negative control cells. Results of 2 independent experiments are shown (C).

Article Snippet: For the small interfering (si) RNA studies, designed and validated siRNA specific for NF-κB p65 (sc-29410), and control non-targeting siRNA (sc-37007) were purchased from Santa Cruz .

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0056399

Figure Lengend Snippet: Representative photomicrographs of human iPS cells: no treatment (A–D), treatment with control siRNA (E–H), treatment wirth p65 siRNA (I–L) (original magnification, x200). A representative EMSA shows NF-κB and AP-1 binding activity in human iPS cells treated with siRNA (M). Representative photographs of alkaline phosphatase staining of human iPS cells treated with siRNA (N–P) (original magnification, x200). No treatment (N), treatment with control siRNA (O), treatment with p65 siRNA (P).

Article Snippet: For the small interfering (si) RNA studies, designed and validated siRNA specific for NF-κB p65 (sc-29410), and control non-targeting siRNA (sc-37007) were purchased from Santa Cruz .

Techniques: Binding Assay, Activity Assay, Staining

Journal: PLoS ONE

Article Title: The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0056399

Figure Lengend Snippet: Western blot analysis of actin, NF-κB p65, IκBα, Oct3/4, Nanog, WT-1, and Pax-2 in human iPS cells treated with siRNA (A). Immunohistochemical staining for NF-κB p65 (B–D) and Oct3/4 (E–G) in human iPS cells treated with siRNA. No treatment (B and E), treatment with control siRNA (C and F), and treatment with p65 siRNA (D and G). Negative control staining without a primary antibody is shown (H) (Original magnification, x200).

Article Snippet: For the small interfering (si) RNA studies, designed and validated siRNA specific for NF-κB p65 (sc-29410), and control non-targeting siRNA (sc-37007) were purchased from Santa Cruz .

Techniques: Western Blot, Immunohistochemical staining, Staining, Negative Control

Journal: Molecular Cancer

Article Title: Herbal compound triptolide synergistically enhanced antitumor activity of amino-terminal fragment of urokinase

doi: 10.1186/1476-4598-12-54

Figure Lengend Snippet: Effects of TPL and ATF on NF-κB and JNK signalling pathway. ( A ) HCT116 cells were treated with TPL and ATF alone or in combination for 24 h. Nuclear proteins were extracted and subjected to Western blotting for p65 detection. Lamin B was used as loading control and α-tubulin was used as a cytoplasmic protein marker to rule out cytoplasmic contamination in the nuclear fraction. ( B ) HCT116 cells were treated with TPL and ATF alone or in combination for 24 h. AKT, p-AKT, and c-FLIP proteins in whole cell lysates were determined with specific antibodies. GAPDH was used as loading control. ( C ) HCT116 cells were transfected with scramble siRNA or NF-κB p65 siRNA. 48 h later, cell extracts were applied for Western blotting analysis using NF-κB p65 and c-FLIP antibodies. ( D ) HCT116 cells were treated with TPL and ATF alone or in combination for 24 h. c-JUN, p-c-JUN and p-JNK protein in whole cell lysates were determined with specific antibodies.

Article Snippet: The siRNA against NF-κB p65 (sc-29410) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Western Blot, Marker, Transfection

Journal: Molecular Cancer

Article Title: Herbal compound triptolide synergistically enhanced antitumor activity of amino-terminal fragment of urokinase

doi: 10.1186/1476-4598-12-54

Figure Lengend Snippet: A working model for the synergistic effect of TPL and ATF on tumour cells. TPL and ATF cooperatively induce apoptosis through caspase-dependent pathway and cell cycle arrest. In HCT116 cells, combined treatment with TPL and ATF at a low dosage inhibits AKT phosphorylation and subsequently inactivates NF-κB. The inhibition of NF-κB leads to down-regulation of c-FLIP, activation of caspases-9/caspase-3 and JNK-c-JUN pathway, and cell cycle arrest, which finally promotes cell apoptosis. In addition, the suppression of NF-κB leads to decreased expression of uPAR, MMP-9 and phospho-FAK, thereby inhibiting tumor cell migration and invasion. Furthermore, ATF can segregate uPA from its receptor uPAR, thus blocking ECM remodeling and anti-angiogenesis processes.

Article Snippet: The siRNA against NF-κB p65 (sc-29410) was purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Techniques: Inhibition, Activation Assay, Expressing, Migration, Blocking Assay

Journal: Mediators of Inflammation

Article Title: lncRNA SNHG14 Plays a Role in Sepsis-Induced Acute Kidney Injury by Regulating miR-93

doi: 10.1155/2021/5318369

Figure Lengend Snippet: Increased expression of SNHG14 in LPS-induced HK-2 cells is due to the activation of the TLR4/NF- κ B pathway. (a) Expression level of SNHG14 in LPS-treated HK-2 cells. (b) Predicted binding site of NF- κ B in the SHNG14 promoter. (c) Activation of the TLR4/NF- κ B pathway in LPS-induced HK-2 cells. (d) ChIP assay showed NF- κ B bound to the SNHG14 promoter. (e) Luciferase assays showed that overexpressing NF- κ B p65 increased the activity of luciferase reporter containing a wild-type SNHG14 promoter. (f) SNHG14 levels in LPS-induced HK-2 cells transfected with NF- κ B p65 overexpression plasmids or shRNAs. Data were shown as mean ± SD, ∗ P < 0.05.

Article Snippet: PcDNA3.1-SNHG14 overexpression (O/E) plasmids, SNHG14 shRNA, pcDNA3.1-NF- κ B p65 O/E plasmids, NF- κ B p65 shRNA (sc-29410-SH, Santa Cruz Biotechnology), miR-93 mimics, and miR-93 inhibitor were transfected into cells using Lipofectamine 3000 (Invitrogen, CA, USA).

Techniques: Expressing, Activation Assay, Binding Assay, Luciferase, Activity Assay, Transfection, Over Expression

Journal: Mediators of Inflammation

Article Title: lncRNA SNHG14 Plays a Role in Sepsis-Induced Acute Kidney Injury by Regulating miR-93

doi: 10.1155/2021/5318369

Figure Lengend Snippet: SNHG14/miR-93 activates NF- κ B and STAT3 signaling through mediating IRAK4 and IL-6R. (a) Predicted binding site of miR-93 in IRAK4 and IL-6R 3′UTR. (b, c) Luciferase assays showed that overexpressing miR-93 decreased the activity of luciferase reporter containing wild-type IRAK4 or IL-6R 3′UTR. (d) IRAK4 and IL-6R protein levels detected by western blot after HK-2 cells were transfected with miR-93 mimics or inhibitor. (e, f) HK-2 cells were transfected with SNHG14 overexpression plasmids and miR-93 mimics, and LPS-induced HK-2 cells were transfected with SNHG14 shRNAs and miR-93 inhibitor; relative proteins in IRAK4/NF-kb and IL-6R/STAT3 signaling were analyzed using western blot. Data were shown as mean ± SD, ∗ P < 0.05.

Article Snippet: PcDNA3.1-SNHG14 overexpression (O/E) plasmids, SNHG14 shRNA, pcDNA3.1-NF- κ B p65 O/E plasmids, NF- κ B p65 shRNA (sc-29410-SH, Santa Cruz Biotechnology), miR-93 mimics, and miR-93 inhibitor were transfected into cells using Lipofectamine 3000 (Invitrogen, CA, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Western Blot, Transfection, Over Expression

Journal: Mediators of Inflammation

Article Title: lncRNA SNHG14 Plays a Role in Sepsis-Induced Acute Kidney Injury by Regulating miR-93

doi: 10.1155/2021/5318369

Figure Lengend Snippet: Silencing SNHG14 alleviates the cellular injury process of IL-1 β and IL-6 in HK-2 cells via miR-93. (a–n) HK-2 cells were treated with IL-1 β or IL-6 and then transfected with SNHG14 shRNAs and miR-93 inhibitor; oxidative stress (a–d), inflammation (e–h), and apoptosis (i–l) were assessed as described above; activation of NF- κ B and STAT3 signaling was tested by detecting relative proteins using western blot (m, n). Data were shown as mean ± SD, ∗ P < 0.05.

Article Snippet: PcDNA3.1-SNHG14 overexpression (O/E) plasmids, SNHG14 shRNA, pcDNA3.1-NF- κ B p65 O/E plasmids, NF- κ B p65 shRNA (sc-29410-SH, Santa Cruz Biotechnology), miR-93 mimics, and miR-93 inhibitor were transfected into cells using Lipofectamine 3000 (Invitrogen, CA, USA).

Techniques: Transfection, Activation Assay, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Triptolide Inhibits Invasion and Tumorigenesis of Hepatocellular Carcinoma MHCC-97H Cells Through NF-κB Signaling

doi: 10.12659/MSM.898801

Figure Lengend Snippet: Effect of TPL on NF-κB activity and NF-κB p65 expression inMHCC-97H cell. ( A ) MHCC-97H cells were treated with 5, 15,or 25 μM TPL for 72 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. ( B ) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. Nuclear extracts were prepared from control and treated cells and subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. ( C ) MHCC-97H cells were treated with 5, 15, or 25 μM TPL for 72 hours. NF-κB p65 protein was detected by western blot assay; ( D ) MHCC-97H cells were treated with 25 μM TPL for 24–60 hours. NF-κB p65 protein was detected by western blot assay.

Article Snippet: NF-κB p65 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, Shanghai, China).

Techniques: Activity Assay, Expressing, Binding Assay, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Triptolide Inhibits Invasion and Tumorigenesis of Hepatocellular Carcinoma MHCC-97H Cells Through NF-κB Signaling

doi: 10.12659/MSM.898801

Figure Lengend Snippet: TPL induces apoptosis by inhibition of NF-κB activity.( A ) The effect of p65 cDNA transfection and TPL treatment on NF-κB DNA-binding activity by EMSA assay. ( B ) Induction of apoptosis by p65 cDNA and TPL in MHCC-97H cells tested by flow cytometry. ( C ) Induction of apoptosis by p65 cDNA and TPL in MHCC-97H cells tested by TUNEL assay. ( D ) the effect of p65 siRNA transfection and TPL on NF-κB DNA-binding activity by EMSA assay. ( E ) Induction of apoptosis by p65 siRNA and TPL in MHCC-97H cells tested by flow cytometry. ( F ) Induction of apoptosis by p65 siRNA and TPL in MHCC-97H cells tested by TUNEL assay. Values are reported as mean ±SD. p <0.05, compared to the control.

Article Snippet: NF-κB p65 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, Shanghai, China).

Techniques: Inhibition, Activity Assay, Transfection, Binding Assay, Flow Cytometry, TUNEL Assay

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Triptolide Inhibits Invasion and Tumorigenesis of Hepatocellular Carcinoma MHCC-97H Cells Through NF-κB Signaling

doi: 10.12659/MSM.898801

Figure Lengend Snippet: TPL inhibits tumor growth and the expression of NF-κB target genes in vivo . MHCC-97H xenografts were generated by inoculating cells subcutaneously (s.c.) in SCID mice. Once transplanted, the cells developed into palpable tumors (about 80 mg), and groups of 10 animals were classified randomly and assigned to different treatment groups. Mice were injected with TPL at 0.4 mg/kg daily for 15 days. The control group received vehicle only. ( A ) TPL retards the growth of MHCC-97H tumor xenografts in nude mice. Tumor volumes in SCID mice were plotted against time; * p <0.05, compared to the control. ( B ) The number of metastatic nodes in each group. Lung tumors were counted with the naked eye. The data represent the mean and the standard deviation (n=10). p values were calculated with the Student’s t-test. * p <0.05. Black is a metastatic node. ( C ) TPL inhibits NF-κB DNA-binding activity in vivo . Tumor xenografts were removed, and nuclear protein extracts were prepared. Binding of NF-κB consensus elements with nuclear extracts was detected by EMSA. ( D ) The expression of NF-κB p65 and MMP-9 was detected by western blotting of tumor tissue extracts. TPL inhibited the expression of NF-κB p65 and MMP-9 in tumor tissues of TPL treated animals compared to controls.

Article Snippet: NF-κB p65 siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, Shanghai, China).

Techniques: Expressing, In Vivo, Generated, Injection, Standard Deviation, Binding Assay, Activity Assay, Western Blot

Journal: PLoS ONE

Article Title: The G32E Functional Variant Reduces Activity of PPARD by Nuclear Export and Post-Translational Modification in Pigs

doi: 10.1371/journal.pone.0075925

Figure Lengend Snippet: PK-15 cells were transfected with pEGFP-C1-wild-type sPPARD (A), and -G32E mutant (B). G32E mutant was treated by leptomycin B (50 nM, 1 h) that blocked CRM1-dependent nuclear export (C). sPPARD subcellular localization was analyzed by GFP fluorescence at 24 hour post-transfection. Cell nuclei were counterstained with DAPI.

Article Snippet: Wild-type sPPARD and G32E mutant were cloned into pEGFP-C1 expression vectors (Addgene), and wild-type sPPARD, G32E, K16R, K17R, K18R and K16-18R mutants were inserted into pcDNA4A-His expression vectors (Addgene).

Techniques: Transfection, Mutagenesis, Fluorescence