Journal: Computational and Structural Biotechnology Journal

Article Title: Severe fever with thrombocytopenia syndrome virus (SFTSV)-host interactome screen identifies viral nucleoprotein-associated host factors as potential antiviral targets

doi: 10.1016/j.csbj.2021.09.034

Figure Lengend Snippet: Characterization of the selected SFTSV inhibitors. (A) Dose-response analyses of artenimol and omacetaxine mepesuccinate are shown, depicting both the antiviral activity (red) and cytotoxicity (black). The grey line indicates 50% of the mock-treated control. The EC 50 , CC 50 , and chemical structure of each drug compound are shown. (B) Co-immunoprecipitation assay was conducted in HEK-293T cell transfected with SFTSV nucleoprotein together with either GPI or RPL3 plasmids. After pull-down, the viral nucleoprotein was detected by anti-HA antibody, while GPI and RPL3 were detected by anti-Myc antibodies. (C) Co-immunoprecipitation assay with the treatment of artenimol or omacetaxine mepesuccinate at the indicated concentrations. (D) Molecular docking analysis predicted the interface between artenimol and GPI. The protein is shown as grey ribbons and the drug compound as color sticks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The plasmids and antibodies, including GPI Human Tagged ORF Clone (RC230292, Origene), RPL3 Human Tagged ORF Clone (RC217987, Origene), mouse anti-HA (Cat #26183; Thermo Fisher), rabbit anti-Myc (Cat #PA1-981, Thermo Fisher), goat anti-mouse-IgG (H + L) HRP (A16072SAMPLE, Thermo Fisher), and goat anti-rabbit-IgG (H + L) HRP (A16104SAMPLE, Thermo Fisher) were purchased from commercial sources.

Techniques: Activity Assay, Control, Co-Immunoprecipitation Assay, Transfection

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Arylsulfatase B Mediates the Sulfonation-Transport Interplay in Human Embryonic Kidney 293 Cells Overexpressing Sulfotransferase 1A3.

doi: 10.1124/dmd.116.070938

Figure Lengend Snippet: Fig. 3. Expression of sulfatases in HEK293 cells. (A) mRNA expression of three sulfatases in HEK293 and SULT293 cells detected by RT-PCR. (B) qPCR measurements of ARSA, ARSB, and ARSC in SULT293 cells. The data were generated from three independent qPCR experiments, with the error bar representing the S.D. (C) Protein expression of ARSA, ARSB, and ARSC in HEK293 and SULT293 cells. WT, wild type.

Article Snippet: Anti-ARSA, anti-ARSB, and anti-ARSC antibodies were purchased from OriGene Technologies (Rockville, MD).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Generated

Journal: Cell reports. Medicine

Article Title: NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy.

doi: 10.1016/j.xcrm.2023.101329

Figure Lengend Snippet: Figure 3. Monocytes from CMML KRASmut patients present a constitutive inflammasome activation (A) Percentage of ASC-specking monocytes in healthy controls (white bars), CMML patients without KRAS mutation (KRASwt, blue bars), and CMML patients with a KRAS mutation (KRASmut, orange bars) at baseline or treated as indicated for NLRP3 (LPS+ATP) or pyrin (LPS+TcdB) inflammasome activation. (B and C) Release of IL-1b from peripheral blood mononuclear cells (PBMCs) (B) and the formation of ASC specks in monocytes (C) in CMML KRASmut patients at baseline or after the indicated stimulation or treatment (LPS+ATP and LPS+TcdB for NLRP3 or pyrin inflammasome, respectively), in the absence/presence of MCC950. (B) Fold increase was calculated to control non-stimulated conditions, where the average of the higher value used to calculate fold increase is 1,066.58 pg/mL. (D) Percentage of extracellular LDH from untreated PBMCs from healthy donors (white bar), KRASwt patients (blue bar), and KRASmut patients (orange bar). Data are normalized to the percentage of monocytes. (E) Plasma concentration of HMGB1 and P2X7 receptor from healthy donors (white bar), KRASwt patients (blue bar), and KRASmut patients (orange bar). For (A)–(E), data are represented as mean ± SEM; each dot represents an individual patient; ordinary one-way ANOVA test (two-tailed) was used for (A) (* compares CMML KRASmut vs. healthy controls; y compares CMML KRASmut vs. CMML KRASwt), and two-tailed t test in (B)–(E). Note that * or yp < 0.05; **p < 0.01; ***p < 0.001; **** or yyyyp < 0.0001; ns, no significant difference (p > 0.05).

Article Snippet: Plasma or cell-free supernatants from fresh EDTA-anticoagulated PB samples from CMML patients and controls were used to quantify the concentration of human IL-1b (#BMS224INST, Invitrogen), human IL-18 (#7620, MBL), human IL-18BP (#EHIL18BP, Invitrogen), human TNF-a (#DTA00D, R&D Systems), human IL-6 (#D6050, R&D Systems), human ASC (#CSB-EL019114HU, Cusabio), human soluble P2X7 receptor (#CSB-EL017325HU, Cusabio), and human HMGB1 (#ARG81185, Arigo Biolaboratories) by ELISA following the manufacturer’s instructions.

Techniques: Activation Assay, Mutagenesis, Control, Clinical Proteomics, Concentration Assay, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: Inter and intra-tumor heterogeneity of paediatric type diffuse high-grade gliomas revealed by single-cell mass cytometry

doi: 10.3389/fonc.2022.1016343

Figure Lengend Snippet: Summary of the 15 antibodies used for the mass cytometry analysis.

Article Snippet: Anti-Human CD49c , 161Dy , Fluidigm , 3161016B , .

Techniques: Mass Cytometry

Journal: Aging Cell

Article Title: Lysosomal storage and impaired autophagy lead to inflammasome activation in G aucher macrophages

doi: 10.1111/acel.12409

Figure Lengend Snippet: Caspase‐1 activation and increased ubiquitinated protein apoptosis‐associated speck‐like proteins ( ASC ) in Gaucher macrophages ( GM s). (A) ELISA or (B) Total supernatants or total protein lysates from treated macrophages from patients (P) and controls (C) were probed for IL ‐1β. (C) Control (C) and GM s (P) were immuno‐stained for LC 3 (red) and NLRP 3 (green) after treatment with lipopolysaccharide ( LPS ) (100 ng) and ATP (5 m m ) for 1 h. Cells were imaged using a confocal microscope (Z‐stack with 0.5 μm thickness). Co‐localization was evaluated using Imaris software. Merged channels are shown as yellow. Pictures represent seven independent experiments performed on cells from seven different patients. Single channels are presented in the supplements. (D) Caspase‐1 was analyzed in total lysate and supernatant by immunoblotting, after treatment with LPS alone or LPS (100 ng)+ ATP (5 m m ) or rapamycin (25 n m ). Data represent five independent experiments. (E) Total protein from control and Gaucher macrophages was immunoblotted for ASC after treatment with LPS (100 ng) and ATP (5 m m ), and then rapamycin (25 n m ), 3 MA (5 m m ) or Baf.1A (10 μ m ) for 40 min. ASC expression was normalized to β‐actin. The blot is representative of seven independent experiments performed on samples from seven different patients and controls. P < 0.05(*) and P < 0.01(**), P < 0.001(***). (F) Control and GM s were immunostained for p62 (red) and ASC (green) in the absence and presence of LPS (100 ng) and ATP (5 m m ) and then imaged by confocal microscopy. Merged channel volumes are shown in yellow, and insets showing surface renderings of three‐dimensional reconstruction using Imaris software are shown in the far right panels.

Article Snippet: Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μ m glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Software, Western Blot, Expressing, Confocal Microscopy

Journal: Aging Cell

Article Title: Lysosomal storage and impaired autophagy lead to inflammasome activation in G aucher macrophages

doi: 10.1111/acel.12409

Figure Lengend Snippet: Inflammasome activation due to impaired autophagy in Gaucher macrophages ( GM s) (1) In both control and GM s, lipopolysaccharide ( LPS ) priming induces activation of p65‐ NF ‐ kB , which is translocated to the nucleus, leading to production of pro‐ IL ‐1β in the cytosol. In control macrophages (1a), LPS stimulates accumulation of ubiquitinated p65‐ NF ‐ kB , which is further recognized by p62, delivered to autophagosomes, and degraded in the lysosome (1a, solid line). In GM s (1b, dashed line), impaired autophagy prevents degradation of p65‐ NF ‐ kB through autophagy and results in its activation (1b). (2) In both control and GM s, stimulation by both LPS and extracellular ATP leads to inflammasome complex formation ( NLRP 3, apoptosis‐associated speck‐like proteins ( ASC ) and caspase1) and activation. (3) Activated inflammasomes undergo ubiquitination of ASC , leading to p62‐mediated engulfment of inflammasomes by autophagosomes. Pro‐ IL ‐1β conversion to active IL ‐1β is limited due to the destruction of activated inflammasomes by autophagolysosomes. In GM s, defective autophagy and lysosomal dysfunction inhibit the elimination of active inflammasomes through autophagy, resulting in the upregulation and secretion of IL ‐1β (3).

Article Snippet: Human macrophages were plated on glass chamber slides and, after various treatments, were fixed with 4% paraformaldehyde for 30 min. For LC3 staining, cells were fixed with cold acetone for 10 min. After one‐step washing with PBS, cells were blocked in PBS containing 0.1% saponin, 100 μ m glycine, and 2% donkey serum (2 h) followed by incubation with antibodies against LC3B (Sigma), SQSTM1/p62 (Cell signaling), ASC (Enzo Life science), cathepsin D (R&D systems), Lamp2 (Hybridoma, Columbia, MD, USA), p65‐NFkB (Origene) for 2 h. Then, cells were washed with PBS three times for 5 min followed by incubation with secondary antibodies.

Techniques: Activation Assay