Journal: International journal of molecular sciences

Article Title: Hemp-Derived Nanovesicles Protect Leaky Gut and Liver Injury in Dextran Sodium Sulfate-Induced Colitis.

doi: 10.3390/ijms23179955

Figure Lengend Snippet: Figure 3. RNVs, SNVs, HSNVs, and LNVs restored the levels of TJ/AJ proteins and attenuated NF-κB activation and oxidative stress markers in the colon of mice with DSS-induced colitis. (A) Comparison of colon lengths. (B) Representative H&E stain of formalin-fixed colon sections for control (CON), 5% dextran sulfate sodium (5% DSS), and 5% DSS+ RNVs, SNVs, HSNVs, and LNVs-exposed mice. (C) The levels of TJ protein (claudin-4, occludin) and AJ protein (E-cadherin, α-tubulin) in the indicated groups are presented. (D,E) The levels of inflammatory marker proteins (p-NF-κB, p-IκB) and oxidative stress marker proteins (COX2, 3-NT, iNOS) in the indicated groups are presented. Densitometric analysis of immunoblots for each protein relative to β-actin is shown. * p < 0.05, ** p < 0.01, *** p < 0.001 between 5% DSS and control groups; # p < 0.05, ## p < 0.01, ### p < 0.001 between 5% DSS vs. RNVs, SNVs, HSNVs, and LNVs groups. Significance of mean values for each group was determined using ANOVA and Tukey’s HSD test. Data represent means ± S.E.M. (n = 5–10/group).

Article Snippet: The primary antibodies used were as follows: 3-Nitrutyrisine (1:3000, Cat. No. ab61392, Abcam, Cambridge, UK), COX-2 (1:1000, Cat. No. SC-376861, Santa Cruz Biotechnology Inc., Dallas, TX, USA), pIκB-α (1:1000, Cat. No. SC-8404, Santa Cruz), p-NF-κB (1:1000, Cat. No. SC-271908, Santa Cruz Biotechnology), occludin (1:1000, Cat. No. SC-271842, Santa Cruz Biotechnology Inc.), E-Cadherin (1:1000, Cat. No. SC-8426, Santa Cruz Biotechnology Inc.), claudin-4 (1:1000, Cat. No. SC-376643, Santa Cruz Biotechnology Inc.), α-tubulin (1:1000, Cat. No. SC-376643, Santa Cruz Biotechnology Inc.), cyclin D1 (1:1000, Cat. No. SC-8396, Santa Cruz Biotechnology Inc.), eNos (1:1000, Cat. No. ab76198, Abcam), and β-actin (1:1000, Cat. No. SC-47778, Santa Cruz Biotechnology Inc.).

Techniques: Activation Assay, Comparison, Staining, Control, Marker, Western Blot

Journal: International journal of molecular sciences

Article Title: Hemp-Derived Nanovesicles Protect Leaky Gut and Liver Injury in Dextran Sodium Sulfate-Induced Colitis.

doi: 10.3390/ijms23179955

Figure Lengend Snippet: Figure 4. RNVs, SNVs, HSNVs, and LNVs restored the levels of TJ/AJ proteins and attenuated NF-κB activation and oxidative stress markers in the small intestine of mice with DSS-induced colitis. (A) Comparison of small intestine lengths. (B) Representative H&E of formalin-fixed small intestine sections for control (CON), 5% DSS, and 5% DSS+ RNVs, SNVs, HSNVs, and LNVs-exposed mice. (C) The levels of TJ protein (claudin-4, occludin) and AJ protein (E-cadherin, α-tubulin) in the indicated groups are presented. (D,E) The levels of inflammatory marker proteins (p-NF-κB, p-IκB) and oxidative stress marker proteins (COX2, 3-NT) in the indicated groups are presented. Densitometric evaluation of immunoblots for each protein relative to β-actin is shown. * p < 0.05, ** p < 0.01, *** p < 0.001 between 5% DSS and control groups; # p < 0.05, ## p < 0.01, ### p < 0.001 between DSS vs. RNVs, SNVs, HSNVs, and LNVs groups. Significance of the mean values for each group was determined using ANOVA and Tukey’s HSD test. Data represent means ± S.E.M. (n = 5–10/group).

Article Snippet: The primary antibodies used were as follows: 3-Nitrutyrisine (1:3000, Cat. No. ab61392, Abcam, Cambridge, UK), COX-2 (1:1000, Cat. No. SC-376861, Santa Cruz Biotechnology Inc., Dallas, TX, USA), pIκB-α (1:1000, Cat. No. SC-8404, Santa Cruz), p-NF-κB (1:1000, Cat. No. SC-271908, Santa Cruz Biotechnology), occludin (1:1000, Cat. No. SC-271842, Santa Cruz Biotechnology Inc.), E-Cadherin (1:1000, Cat. No. SC-8426, Santa Cruz Biotechnology Inc.), claudin-4 (1:1000, Cat. No. SC-376643, Santa Cruz Biotechnology Inc.), α-tubulin (1:1000, Cat. No. SC-376643, Santa Cruz Biotechnology Inc.), cyclin D1 (1:1000, Cat. No. SC-8396, Santa Cruz Biotechnology Inc.), eNos (1:1000, Cat. No. ab76198, Abcam), and β-actin (1:1000, Cat. No. SC-47778, Santa Cruz Biotechnology Inc.).

Techniques: Activation Assay, Comparison, Control, Marker, Western Blot

Journal: The Journal of Cell Biology

Article Title: VHL promotes immune response against renal cell carcinoma via NF-κB–dependent regulation of VCAM-1

doi: 10.1083/jcb.201608024

Figure Lengend Snippet: VHL and hypoxia affect noncanonical NF-κB pathways in ccRCC cells. (A and B) Western blots of RCC4-VHL–positive or –negative cells under normoxia (Nx) or hypoxia 0.5% O 2 (Hp); A) or cells transfected with a scrambled siRNA (Scr) or siRNAs specific for IKKα, NIK, p52, or p50 (B). Images show protein levels of VCAM-1, NIK, IKKα, Rel B, p65, p52, p50, VHL, and α-Tubulin as a loading control. A representative Western blots are shown. n = 3.

Article Snippet: siRNA experiments were performed with specific pools of siRNAs directed against human VCAM-1 (sc-29519), HIF-1α (sc-35561), HIF-2α (sc-35316), vhl (sc-36816), phd1 (sc-45616), phd2 (sc-45537), phd3 (sc-45799), nik (sc-36065), ikkα (sc-29365), p52 (sc-29409), and p50 (sc-29407) or with a nontargeted pool of control scrambled siRNAs (sc-37007) from Santa Cruz Biotechnology, Inc.

Techniques: Western Blot, Transfection, Control

Journal: Cancer Biology & Therapy

Article Title: The NFκB inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype

doi: 10.4161/cbt.28158

Figure Lengend Snippet: Figure 3. Effects of silencing of NFκB/p50 expression on differentiation of GSCs. (A) GSC11 cells were dissociated, suspended in serum-free DMEM medium in the absence of B-27, EGF, and β-FGF, and then were infected with an NFκB/p50-targeted shRNA lentiviral particles for 48 h in the presence of polybrene (5 μg/mL). Forty-eight hours later, (A) the cells were observed under a phase contrast microscopy (200× magnification). **P < 0.01, shNFκB/p50 vs. shNT control; (B) cell lysates were prepared and subjected to western blot analysis of CD133, Sox2, MAP-2, and p50. β-actin was used as a loading control.

Article Snippet: GSC11 cells were dissociated, plated into 6-well plates, and then infected with an NFκB/p50-targeted shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) in the presence of 5 μg/mL of polybrene (Sigma-Aldrich) for 24 h. The cells were then selected with puromycin.

Techniques: Expressing, Infection, shRNA, Microscopy, Control, Western Blot

Journal: Cancer Biology & Therapy

Article Title: The NFκB inhibitor, SN50, induces differentiation of glioma stem cells and suppresses their oncogenic phenotype

doi: 10.4161/cbt.28158

Figure Lengend Snippet: Figure 3. Effects of silencing of NFκB/p50 expression on differentiation of GSCs. (A) GSC11 cells were dissociated, suspended in serum-free DMEM medium in the absence of B-27, EGF, and β-FGF, and then were infected with an NFκB/p50-targeted shRNA lentiviral particles for 48 h in the presence of polybrene (5 μg/mL). Forty-eight hours later, (A) the cells were observed under a phase contrast microscopy (200× magnification). **P < 0.01, shNFκB/p50 vs. shNT control; (B) cell lysates were prepared and subjected to western blot analysis of CD133, Sox2, MAP-2, and p50. β-actin was used as a loading control.

Article Snippet: GSC11 cells were dissociated, plated into 6-well plates, and then infected with an NFκB/p50-targeted shRNA lentiviral particles (Santa Cruz Biotechnology, Inc.) in the presence of 5 μg/mL of polybrene (Sigma-Aldrich) for 24 h. The cells were then selected with puromycin.

Techniques: Expressing, Infection, shRNA, Microscopy, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Chlamydia abortus Pmp18.1 Induces IL-1β Secretion by TLR4 Activation through the MyD88, NF-κB, and Caspase-1 Signaling Pathways

doi: 10.3389/fcimb.2017.00514

Figure Lengend Snippet: Pmp18.1 activates MyD88, NFκB and Caspase-1 expression in BMDCs. Cell lysates were prepared from BMDCs incubated with Pmp18.1 (10 μg/ml) for 1, 2, or 24 h. Protein extracts (40 μg/well) were separated by SDS-PAGE and detected by immunoblotting analysis incorporating the Pierce™ ECL Western Blotting Substrate using antibodies to the corresponding proteins. The proteins bands were visualized using the ImageQuant LAS-4000 imaging system. Protein levels, normalized to GAPDH, were then quantified using Image J software. The data shows the expression levels and fold induction of TLR4 (A) , MyD88 (B) , NF-κB p50 (C) , and Caspase-1 (D) . Each experiment was repeated at least two times with similar findings. For NF-κB p65 nuclear migration, BMDCs were incubated for 24 h with rPmp18.1 and nuclear NF-κB p65 was detected by confocal microscopy using a FITC conjugated monoclonal antibody to NF-κB p65 (clone D14E12, Cell Signaling Technology). (E) NF-κB p65 unit was stained in green, and nuclei were visualized by DAPI counterstain (blue). Note the diffuse distribution of NF-κB p65 (green) in the nucleus. The statistical significance difference between two groups was evaluated by Student's t -test and between more than two groups by the one-way analysis of variance (ANOVA). Differences were considered to be statistically significant at * p < 0.05.

Article Snippet: Membranes were blocked with 5% non-fat dry milk solution (catalog number 9999) (Cell Signaling Technology, Danvers, MA) at room temperature for 1 h and reacted with the corresponding protein antibodies at 4°C overnight: myeloid differentiation primary response gene 88 (MyD88) (HFL-296, polyclonal; catalog number sc-11356), nuclear factor kappa B (NF-κB) p50 (H-119, polyclonal; catalog number sc-7178), NFκB p65 (F-6, monoclonal; catalog number sc-8008), Caspase-1 (14F468, monoclonal; catalog number sc-56036), GAPDH (FL-335, polyclonal; catalog number sc-25778) (Santa Cruz, Dallas, TX).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Imaging, Software, Migration, Confocal Microscopy, Staining