Journal: Journal of Virology

Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na + , K + /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

doi: 10.1128/JVI.01861-17

Figure Lengend Snippet: AICAR antagonizes HCMV inhibition by digitoxin in conjunction with reduced AMPK phosphorylation and autophagy. (A) HFFs were infected with HCMV (100 PFU/well), followed by treatment with compounds at the indicated doses. (Left) Digitoxin plus AICAR; (center) digitoxin plus CC; (right) digitoxin plus GCV. The effects of drug combinations were analyzed by WB at 24 hpi (B) and 72 hpi (C) for LC3-II, pAMPK, AMPK, UL44, and pp65. (D) Control and ATG5 KD cells were infected with HCMV (MOI = 1) and treated with AICAR (0.8 mM), digitoxin (30 nM), or GCV (5 μM). Lysates were prepared to detect p62 and viral pp65. β-Actin was used as a loading control. Blots are representative of three independent experiments.

Article Snippet: Antibodies for detection of HCMV proteins were as follows: mouse monoclonal anti-pp65 (Vector Laboratories, Burlingame, CA), mouse anti-IE1 and -IE2 (MAb810; Millipore, Billerica, MA), and mouse monoclonal anti-UL44 (Santa Cruz Biotechnology).

Techniques: Inhibition, Infection

Journal: Journal of Virology

Article Title: Digitoxin Suppresses Human Cytomegalovirus Replication via Na + , K + /ATPase α1 Subunit-Dependent AMP-Activated Protein Kinase and Autophagy Activation

doi: 10.1128/JVI.01861-17

Figure Lengend Snippet: Digitoxin fails to induce autophagy or to inhibit HCMV in α1 KD cells. (A) Control and α1 KD cells were infected and treated with digitoxin (30 nM), AICAR (0.8 μM), digitoxin plus AICAR, or GCV. Levels of pp65, pAMPK, and p62 were detected by WB at 72 hpi. (B) Uninfected cells were treated with digitoxin or GCV. Levels of pAMPK and p62 were detected by WB. (C) α1 KD and control HFFs were infected with HCMV Towne (100 plaques/well), followed by treatment with digitoxin or GCV. Plaques were counted under each condition at day 8 postinfection. (D) (Left) Lysates from panel A were used to detect mTOR, p-mTOR, and the mTOR substrates p-Ser S6K and p-Thr S6K. (Right) p-mTOR levels were quantitated by densitometry. (E) pLKO.1 and α1 KD cells were infected and treated with digitoxin or GCV for 12 h. One-milligram aliquots of protein from the prepared lysates were used to pull down ULK1. AMPK, mTOR, and α1 were detected by WB of the input samples.

Article Snippet: Antibodies for detection of HCMV proteins were as follows: mouse monoclonal anti-pp65 (Vector Laboratories, Burlingame, CA), mouse anti-IE1 and -IE2 (MAb810; Millipore, Billerica, MA), and mouse monoclonal anti-UL44 (Santa Cruz Biotechnology).

Techniques: Infection

Journal: International journal of molecular sciences

Article Title: WGA-M001, a Mixture of Total Extracts of Tagetes erecta and Ocimum basilicum , Synergistically Alleviates Cartilage Destruction by Inhibiting ERK and NF-κB Signaling.

doi: 10.3390/ijms242417459

Figure Lengend Snippet: Figure 4. WGA-M001 regulated OA-related molecules through dephosphorylation in the NF-κB and ERK pathways. For in silico analysis, 200 µg/mL of Tagetes erecta, Ocimum basilicum, or WGA-M001 was used for treatment of chondrocytes for 12 h along with IL-1β (1 ng/mL), and RNA sequencing was performed. (A) In each signaling pathway, the number of genes upregulated by IL-1β and then downregulated by T. erecta, O. basilicum, or WGA-M001 was shown. (B,C) Protein levels of pp38, p38, pJNK, JNK, pERK, ERK, pp65, p65, and IκB were detected by Western blot analysis and relative intensities were quantified by densitometry (n = 5). Each protein level was normalized to ERK. Data are represented as mean ± SD as results of analysis by one-way ANOVA with Dunnett’s multiple comparisons test (n = 5). * p < 0.05, **** p < 0.0001, and n.s = not significant.

Article Snippet: 2023, 24, 17459 12 of 16 cam), rabbit anti-COX-2 (ab52237; Abcam), mouse anti-IκB (9242; Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-p65 (#6956; CST), mouse anti-pp65 (#13346; CST), mouse anti-p38 (#9212; CST), mouse anti-pp38 (#9215S; CST), mouse anti-c-JNK (#9252S; CST), mouse anti-pJNK (#9251S; CST), and mouse anti-pERK (#9101S; CST).

Techniques: De-Phosphorylation Assay, In Silico, RNA Sequencing, Western Blot

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Human Cytomegalovirus Inhibition by Cardiac Glycosides: Evidence for Involvement of the hERG Gene

doi: 10.1128/AAC.00898-12

Figure Lengend Snippet: CG inhibit the expression of HCMV genes and NF-κB. (A) HFFs were infected and treated with the indicated concentrations of digoxin, ouabain, or GCV (10 μM). The expression of immediate-early (IE1 and IE2), early (UL84 and UL44), and late HCMV proteins (pp65) was determined in cell lysates after 72 h. (B) HFF cells were infected with HCMV, treated with digoxin (0.1 μM), ouabain (0.05 μM), or GCV (10 μM), and the expression levels of IE1 and IE2 proteins were determined at the indicated time points. (C) HFF cells were infected with HCMV and treated with DMSO (control), digoxin, or ouabain at the indicated concentrations. The concentration of GCV was 10 μM. The levels of NF-κB (p65 subunit) protein were determined after 12 h in total cell lysates. β-Actin was used as a sample loading control.

Article Snippet: The following antibodies were used: mouse anti-IE1 and -IE2 antibody (MAb810), mouse anti-β-actin antibody (Millipore, Billerica, MA), mouse anti-UL84 antibody, mouse anti-NF-κB antibody (p65), mouse anti-UL44 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-pp65 antibody (Vector Laboratories, Burlingame, CA), HRP-conjugated anti-rabbit IgG antibody (Cell Signaling Technology, Beverly, MA), rabbit anti-KV11.1 (hERG) antibody (Alomone Labs, Jerusalem, Israel), rabbit anti-GFP antibody (G1544; Sigma), and HRP-conjugated anti-mouse IgG (GE Healthcare, Waukesha, WI).

Techniques: Expressing, Infection, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Inhibition of IL1R1 or CASP4 attenuates spinal cord injury through ameliorating NLRP3 inflammasome-induced pyroptosis

doi: 10.3389/fimmu.2022.963582

Figure Lengend Snippet: Histopathological changes. (A) HE staining demonstrated that the structure of the injured spinal cord tissue became clearer after treatment, with fewer cavities and necroses as compared with SCI group (Scale bars = 100μm, the arrows point to cavities and necroses), and the expression level of the biomarkers in the spinal cord was reduced (Scale bars = 20μm) (n = 3 rats per group, *p < 0.05, **p < 0.01, ***p < 0.001, t-test). (B) Representative images showing pp65 and DAPI co-staining of the spinal cord sections from the rats of the four groups (Scale bars = 20μm) (n = 3 rats per group, *p < 0.05, **p < 0.01, ***p < 0.001, t-test).

Article Snippet: The antibodies and reagents used in this study included anti-IL1R1 (Abclonal, A5727), anti-GAPDH (Proteintech, HRP-60004), anti-CASP4 (Affinity, AF5130), anti-cleaved CASP4 (Affinity, AF5373), anti-IGSF6 (Abclonal, A15128), anti-phospho-NF-κB pp65 (Servicebio, GB13025), anti-Bcl2 (Abclonal, A0208), anti-CASP3 (Abclonal, A2156), anti-IL1B (Abclonal, A16288), anti-NLRP3 (Affinity, DF7438), anti-ASC (Abcam, ab180799), anti-GSDMD (Cell signaling technology, 39754), anti-cleaved GSDMD (Cell signaling technology, 10137), anti-NF-κB p65 (Cell signaling technology, 3033), Goat anti-rabbit IgG-HRP (Proteintech, HRP60004), Goat anti-rabbit pp65 (Servicebio, GB21303), TUNEL (Servicebio, GB1501), DAPI (Servicebio, G1012), Anakinra (MCE, AMG-719), and Belnacasan (Selllock, also known VX-765, S2228).

Techniques: Staining, Expressing