Journal: Cell & Bioscience

Article Title: SNX27-driven membrane localisation of OTULIN antagonises linear ubiquitination and NF-κB signalling activation

doi: 10.1186/s13578-021-00659-5

Figure Lengend Snippet: SNX27 interacts with Met1 via OTULIN. A SNX27 is selectively enriched by Met1 linkage. Bar plot showed enrichment of SNX27 by di-ubiquitin linkages in mESC and NPC. The raw data was obtained from previous publication . B Streptavidin based immunoprecipitation assay to validate the interaction between SNX27 and di-ubiquitin linkages. Biotin-tagged di-ubiquitin coupled to streptavidin beads were incubated with HeLa whole cell lysate. SNX27 and OTULIN antibodies were used to detect the interaction after immunoblotting. Immunoblotting was performed at least twice, and one representative figure was shown. C Streptavidin based immunoprecipitation assay to validate the interaction between SNX27 and Met1 linkage in HeLa and mES cells. Biotin-tagged K48, K63 and Met1 di-ubiquitin coupled to streptavidin beads were incubated with HeLa or mES whole cell lysate. SNX27 antibody were used to detect the interaction after immunoblotting. Black triangle indicates specific signal. D Interaction proteomics analysis of SNX27. HeLa cells expressing control vector or Flag-SNX27 were lysed and incubated with Flag-M2 beads, after which protein on-bead digestion and peptide desalting were performed before mass spectrometry analysis. Data were analysed using DEP package as described . OTULIN and SNX27 are highlighted. Full dataset is available in Additional file : Table S1. E OTULIN mediates the interaction of SNX27 and Met1 linkage in vitro. Bacterial recombinant expressed GST or GST-SNX27 were incubated with Met1 di-ubiquitin in the presence or absence of Flag-OTULIN. Indicated antibodies were used to detect the protein in input and pull-down samples. F OTULIN is required for SNX27 and Met1 linkage interaction in vivo. HeLa cells with inducible shRNA-mediated knockdown for luciferase or OTULIN were used to exam the interaction between SNX27 and Met1 linkage. Cells were treated with doxycycline for indicated time. Immunoprecipitation and immunoblotting assays were performed as B

Article Snippet: Antibodies used were: SNX27 (Abcam, ab77799 and Beythl, A305-439A-T), OTULIN (Cell Signaling, 14127), Flag (Sigma, F1804), Myc (Proteintech, 16286–1-AP), HA (Sigma, H6908), GAPDH (GeneTex, GTX100118), GST (Thermo, PA1-982A), GFP (Santacruz, SC-9996 and Proteintech, 50,430–2-AP), Streptavidin-HRP (Thermo, 21,130), p65 (Cell Signaling, 8242), phosphor-p65 (Cell Signaling, 3033), IκBα (Cell Signaling, 4814), phosphor-IκBα (Cell Signaling, 2859), phosphor-IKKα/β (Cell Signaling, 2697), TNFR1 (Proteintech, 21574), linear ubiquitin (MABS451, Millipore), RIPK1 (Cell Signaling, 3493) and ATP1A1 (Abcam, ab7671).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Incubation, Western Blot, Expressing, Control, Plasmid Preparation, Mass Spectrometry, In Vitro, Recombinant, In Vivo, shRNA, Knockdown, Luciferase

Journal: Scientific Reports

Article Title: A membrane transporter determines the spectrum of activity of a potent platinum–acridine hybrid anticancer agent

doi: 10.1038/s41598-020-72099-z

Figure Lengend Snippet: Pyrimethamine (PM) protects A549 lung adenocarcinoma cells from the cytotoxic effects of compound 1 by blocking its cellular uptake. ( a) PM, a high-affinity, selective inhibitor of hMATE1. ( b) Confocal fluorescence microscopy images of A549 cells treated for 4 h with 10 µM compound 1 with or without PM. Scale bars: 20 µm. Acridine fluorescence in the blue channel is displayed in cyan. ( c) Mean fluorescence intensities in the acridine channel (arbitrary units) of > 100 selected A549 cells (treated according to the conditions in panel (b) determined in 6 images from 2 independent experiments; ****, p < 10 –4 , mean ± S.D., two-tailed t-test with unequal variance. ( d) Accumulation of compound 1 (100 nM, 4 h) in A549 cells in the absence and presence of PM (100 nM) determined by inductively coupled mass spectrometry, ICP-MS), data are the mean of three independent experiments ± S.E.M, two-tailed t-test. ( e) Cytoprotective effect of PM-mediated inhibition of uptake of compound 1 into A549 cells monitored by a cell proliferation assay. Data are the mean of two experiments performed in sextuplicate ( n = 12) ± S.E.M; ***, p < 0.001, one-way ANOVA with post hoc test.

Article Snippet: The human cell lines, A549 (lung adenocarcinoma, doubling time 21 h) and HCT-116 (colorectal carcinoma, doubling time 17 h) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Blocking Assay, Fluorescence, Microscopy, Two Tailed Test, Mass Spectrometry, Inhibition, Proliferation Assay

Journal: Scientific Reports

Article Title: A membrane transporter determines the spectrum of activity of a potent platinum–acridine hybrid anticancer agent

doi: 10.1038/s41598-020-72099-z

Figure Lengend Snippet: Transient knockdown of the membrane transporter hMATE1 ( SLC47A1 ) attenuates uptake and cytotoxicity of compound 1 . ( a ) Western blot analysis of hMATE1 and GAPDH (loading control) protein levels in A549 cells reverse-transfected with scrambled RNA sequence (“mock”) (left) or hMATE1 siRNA (right) (one 72-h transfection at 2.5 nM siRNA). Full-length blots are presented in Supplementary Fig. . ( b ) Immunofluorescence staining of fixed, permeabilized A549 cells 72 h after siRNA knockdown or mock treatment. Scale bars: 20 µm. ( c ) Uptake of compound 1 into A549 cells after siRNA or mock transfection determined by ICP-MS. Accumulated platinum (ng/10 6 cells) is shown as the mean ± S.E.M. of three independent experiments. The assay was performed several times under slightly varied conditions with similar results (see Supplementary Fig. ); p < 0.01, **). ( d ) Effect of hMATE1 knockdown on the cytotoxicity of compound 1 in A549 cells assessed by a cell proliferation assay (MTS). Data are the mean ± S.E.M of two independent experiments performed in triplicate ( n = 6; the results were significant at p < 0.05 (*) and p < 0.001 (***), respectively; two-tailed t-test). For additional data and replicates, see Supplementary Fig. .

Article Snippet: The human cell lines, A549 (lung adenocarcinoma, doubling time 21 h) and HCT-116 (colorectal carcinoma, doubling time 17 h) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Knockdown, Membrane, Western Blot, Control, Transfection, Sequencing, Immunofluorescence, Staining, Proliferation Assay, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Construction, purification, and characterization of FGF21-164. ( A ) FGF21-164 sequence and FGF21 sequence. In the FGF21-164 sequence, red marked the mutated amino acid and blue represents the TR acid sequence from hCD164. In the FGF21 sequence, the red-labeled amino acid represents the amino acid sequence whose n-terminal is deleted in FGF21-164. ( B ) Subcloned cell culture supernatants expressing FGF21-164 and FGF21 were analyzed using Western blotting (Lane M: molecular weight marker. Lane 1~7: FGF21-164 clones 1, 2, 3, A4, B2, F3, and H4. Line 8: FGF21 clone2). ( C ) The purified FGF21-164 and FGF21 were assayed using SDS-PAGE and Western blotting (Lane M: molecular weight marker. Lane 1: purified FGF21-164. Line 2: purified FGF21). ( D ) Purity and hydrodynamic radius of FGF21-164 were analyzed using SEC-HPLC. ( E ) MALDI-TOF mass-spectrometry analysis of FGF21-164. ([M + H] + denotes the singly charged ionic forms, and [M + 2H] 2+ denotes doubly charged ionic forms). ( F ) Circular Dichroism (CD) spectra of FGF21-164 and FGF21.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Purification, Sequencing, Labeling, Cell Culture, Expressing, Western Blot, Molecular Weight, Marker, Clone Assay, SDS Page, Mass Spectrometry, Circular Dichroism

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Glycan forms analysis of the protein FGF21-164. ( A ) O-linked glycan forms of FGF21-164. ( B ) N-linked glycan forms of FGF21-164.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Effect of FGF21-164 in adipocytes induced by 3T3-L1. ( A ) The process of inducing 3T3-L1’s differentiation into adipocytes. ( B ) Reduction in glucose in medium stimulated by FGF21-164 in adipocytes induced by 3T3-L1. Data are means ± SEM (n = 6). * p < 0.05, *** p < 0.001, and **** p < 0.0001 compared to 0 μg/mL FGF21-164. ( C ) Phospho-Erk1/2-specific bands (Thr202/Tyr204; 44–42 kDa) were detected in 3T3-L1-derived adipocytes after FGF21-164 stimulation. ( D ) Evaluation of lipid droplet accumulation in adipocytes induced by 3T3-L1 with or without FGF21-164. The scale bar represents 50 μm.Data are the means ± SEM (n = 6). * p < 0.05, **** p < 0.0001.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Pharmacokinetics of FGF21-164. FGF21-164 plasma levels in C57BL/6 mice as determined via targeted LC-MS after i.v. (tail vein) bolus injection of FGF21-164 protein.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Liquid Chromatography with Mass Spectroscopy, Injection

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: The results of an oral glucose tolerance test applied to ob / ob mice after a single FGF21-164 treatment. ( A ) Blood glucose levels were measured at 0, 2, 3, 4, 5, 6, 7, and 8 h after the administration of FGF21-164. Data are the means ± SEM (n = 4–5). * p < 0.05 and ** p < 0.01 compared with the model. ( B ) The decrease in blood glucose levels post-administration relative to the baseline measurement at 0 h. Data are the means ± SEM (n = 4–5). * p < 0.05 compared with the model.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Effect of a single administration of FGF21-164 on ob / ob mice. ( A , D , G ) Fasting glucose levels. Blood glucose levels were measured at 7, 21, and 28 days after a single dose of FGF21-164 was administered. Data are the means ± SEM (n = 4–5). ns: no significance. * p < 0.05, ** p < 0.01, and **** p < 0.0001. ( B , E , H ) The OGTT at 7, 21, and 28 days. After oral glucose administration, blood glucose levels were measured. Data are the means ± SEM (n = 4–5). * p < 0.05, *** p < 0.001, and **** p < 0.0001 at 6 mg·kg −1 vs. model. # p < 0.05, ## p < 0.01 and #### p < 0.0001 at 12 mg·kg −1 vs. model. ( C , F , I ) The glucose AUC during the OGTT process. Data are the means ± SEM (n = 4–5). ns: no significance. * p < 0.05, *** p < 0.001. ( J ) Body weight measured at 0, 7, 21, and 28 days. Data are the means ± SEM (n = 4–5). ns: no significance.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Effect of repeated administration of FGF21-164. ( A ) Body weight changes. The DIO mice were treated with PBS or FGF21-164 once every 2 days. Data are the means ± SEM (n = 5–7). **** p < 0.0001 vs. Ctrl. #### p < 0.0001 for FGF21-164 vs. the model. ( B ) The liver sections were stained with Oil-red-O. The scale bar represents 100 μm. DIO mice showed many red lipid droplets in hepatic tissue (indicated by the black arrow), while lipid droplet levels were lower after the FGF21-164 treatment.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Staining