Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Overview of all differentially expressed genes categorized according to their activity in cancer biology.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: Single-Molecule RNA Sequencing Reveals IFNγ-Induced Differential Expression of Immune Escape Genes in Merkel Cell Polyomavirus–Positive MCC Cell Lines

doi: 10.3389/fmicb.2021.785662

Figure Lengend Snippet: Validation of differential gene expression in MCC cell lines on protein level and mRNA level. (A) TPM values were acquired from our sequencing output. The data represent mean values ± SEM from three independent experiments (two for MKL-1 control). (B) After 72 h of treatment with or without IFNγ (3,000 U/ml), the Merkel cell carcinoma cell lines MKL-1, MKL-2, and WaGa were stained for signal transducer and activator of transcription 1-alpha/beta (STAT1), bone marrow stromal antigen 2 (BST2), C-X-C motif chemokine 10 (CXCL10), and HLA class II histocompatibility antigen gamma chain (CD74) and analyzed by flow cytometry. The average MFI of three independent experiments ± standard error is indicated. p -values were determined using the paired student’s t -test. * p < 0.05; ** p < 0.01; ns, not significant.

Article Snippet: For antibody staining, 1 × 10 6 cells of each MCC cell line were cultured in a six-well plate in the presence or absence of IFNγ (3,000 U/ml) for 72 h. Cells were washed with fluorescence-activated cell sorting (FACS) buffer (dulbecco’s phosphate-buffered saline with 1% FBS and 0.2% sodium azide) and stained for the following markers: STAT1α/β, Indoleamine-2,3-dioxygenase (IDO), C-X-C motif chemokine 10 (CXCL10), PD-L1, bone marrow stromal antigen 2 (BST2), HLA class II histocompatibility antigen gamma chain (CD74), and HLA A, B, and C (HLA-ABC) with the following monoclonal antibodies: anti-human STAT1 (phycoerythine; PE, clone REA272, Miltenyi Biotec), anti-IDO1 (PE, clone eyedio, Invitrogen), anti-human CXCL10 (PE, clone REA334, Miltenyi Biotec), anti-CD274 (Pe-Cy7, clone MIH1, BD Biosciences), anti-human BST2 (PE, clone REA202, Miltenyi Biotec), anti-CD74 (PE, clone 5-329, Miltenyi Biotec), and anti–HLA-ABC (fluorescein-5-isothiocyanat, BD Biosciences).

Techniques: Biomarker Discovery, Gene Expression, Sequencing, Control, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: The anti-hyperglycemic efficacy of a lipid-lowering drug Daming capsule and the underlying signaling mechanisms in a rat model of diabetes mellitus

doi: 10.1038/srep34284

Figure Lengend Snippet: ( A ) Images (×400) of immunofluorescence staining of GLP-1 (glucagon-like peptide-1) and GIP (gastric inhibitory polypeptide) expression in ileum in the Control, DM and DMC groups. ( B ) Immunofluorescence results (×400) indicating GIP expression. (scale bar: 20μm). ( C ) Quantification of the GLP-1 fluorescence intensity (integrated density per stained area). ( D ) Quantification of the GIP fluorescence intensity (integrated density per stained area). *p < 0.05 vs Control, # p < 0.05 vs DM; mean ± SEM.

Article Snippet: The ileum tissues were fixed with 4% buffered paraformaldehyde and were incubated with GLP-1 and GIP antibody (Wuhan Boster Biological Technology., LTD, China) at 4 °C overnight, followed by incubation with the secondary antibody anti-rabbit IgG (Alexa Fluor 488: Molecular Probes, Eugene, OR).

Techniques: Immunofluorescence, Staining, Expressing, Control, Fluorescence

Journal: Scientific Reports

Article Title: The anti-hyperglycemic efficacy of a lipid-lowering drug Daming capsule and the underlying signaling mechanisms in a rat model of diabetes mellitus

doi: 10.1038/srep34284

Figure Lengend Snippet: ( A ) GLP-1 concentrations in the serum in the in the Control, DM and DMC groups. ( B ) GCG (the gene encoding GLP-1) mRNA levels in ileum. ( C ) GIP concentrations in the serum in the five groups. ( D ) GIP mRNA levels in ileum. ( E ) Insulin levels in the serum in the five groups. *p < 0.05, **p < 0.01 vs Control, # p < 0.05, ## p < 0.01 vs DM; mean ± SEM.

Article Snippet: The ileum tissues were fixed with 4% buffered paraformaldehyde and were incubated with GLP-1 and GIP antibody (Wuhan Boster Biological Technology., LTD, China) at 4 °C overnight, followed by incubation with the secondary antibody anti-rabbit IgG (Alexa Fluor 488: Molecular Probes, Eugene, OR).

Techniques: Control

Journal: Scientific Reports

Article Title: The anti-hyperglycemic efficacy of a lipid-lowering drug Daming capsule and the underlying signaling mechanisms in a rat model of diabetes mellitus

doi: 10.1038/srep34284

Figure Lengend Snippet: ( A ) Molecular structure of Emodin. ( B ) Relative protein levels of p-GSK-3β, GSK-3β, β-catenin and TCF7L2 in Control and Emodin groups under different glucose conditions, detected by the western blot analysis. ( C ) Relative mRNA expression of β-catenin and TCF7L2 in Control and Emodin groups under different glucose conditions, detected by real-time PCR. ( D ) GLP-1 and GIP concentration measured by ELISA in the cell supernatant in Control and Emodin groups under different glucose conditions. *p < 0.05, **p < 0.01 vs control under no glucose, # p < 0.05, ## p < 0.01 vs control under low glucose, &p < 0.05, && p < 0.01 vs control under high glucose; mean ± SEM. ( E,F ) Inhibition of TCF7L2 and GSK-3β abolished DMC’s effects. ( E ) Effects of emodin (the main component of DMC) and SB216763 (a specific GSK-3β inhibitor) on the protein levels of β-catenin and TCF7L2 under HG (high glucose) conditions. ( F ) Relative mRNA expression of β-catenin and TCF7L2 in the three groups. ( G ) Relative protein expression of TCF7L2 in HG, HG + emodin and HG + emodin + TCF7L2 SiRNA groups. ( H ) GLP-1 and GIP concentration measured by ELISA in the cell supernatant in the three groups. *p < 0.05, **p < 0.01 vs HG, #p < 0.05, ##p < 0.01 vs HG + Emodin; mean ± SEM.

Article Snippet: The ileum tissues were fixed with 4% buffered paraformaldehyde and were incubated with GLP-1 and GIP antibody (Wuhan Boster Biological Technology., LTD, China) at 4 °C overnight, followed by incubation with the secondary antibody anti-rabbit IgG (Alexa Fluor 488: Molecular Probes, Eugene, OR).

Techniques: Control, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Scientific Reports

Article Title: The anti-hyperglycemic efficacy of a lipid-lowering drug Daming capsule and the underlying signaling mechanisms in a rat model of diabetes mellitus

doi: 10.1038/srep34284

Figure Lengend Snippet: DMC is an effective strategy to lower blood glucose by upregulating the GLP-1 and GIP secretion and increase the GCG, GIP transcription by enhancing the TCF7L2 expression through the GSK-3β/β-catenin pathway.

Article Snippet: The ileum tissues were fixed with 4% buffered paraformaldehyde and were incubated with GLP-1 and GIP antibody (Wuhan Boster Biological Technology., LTD, China) at 4 °C overnight, followed by incubation with the secondary antibody anti-rabbit IgG (Alexa Fluor 488: Molecular Probes, Eugene, OR).

Techniques: Expressing

Journal: Scientific Reports

Article Title: GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

doi: 10.1038/s41598-021-93862-w

Figure Lengend Snippet: Cellular responses to GIP_HUMAN[22–51]. ( a, b ) Confocal laser-scanning microscopy images of the fluorescent GIP_HUMAN[22–51] peptides bound to cultured cells. Growing HAoECs ( a ) or THP1-derived macrophages deprived of serum for 16 h ( b ) were overlaid without (left panels) or with 10 –6 M FAM-GIP_HUMAN[22–51] (right panels) for 30 or 5 min, respectively. Cells were washed, fixed, nuclei counterstained with DAPI (blue) and the cell surface-bound green fluorescence visualised. Scale bar represents 50 μm. ( c, d ) Nuclear translocation of NF-κB. HAoECs ( c ) or THP1-derived macrophages ( d ) were incubated without (left panel) or with 10 –6 M GIP_HUMAN[22–51] for 60 min, and immunocytochemical staining was performed using an NF-κB p65 subunit antibody to detect its nuclear translocation. ( e, f ) Degradation of IκB-α. HAoECs ( e ) or THP1-derived macrophages ( f ) were stimulated with 10 –7 M GIP_HUMAN[22–51] for the indicated times and subjected to western blot analysis using the anti-IκB-α antibody to assess the time-course of IκB-α degradation. The panels show the cropped blots and the full-length blots are presented in the Supplementary Information. ( g–i ) Upregulation of MMP8 gene expression by GIP_HUMAN[22–51]. HAoECs were incubated with 10 –7 M GIP_HUMAN[22–51] for the indicated times ( g ) or with indicated doses for 48 h ( h ), and MMP8 mRNA and β-actin levels were quantified. Data represent the fold changes (mean ± S.E.M) of MMP8 mRNA copies relative to β-actin mRNA ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with vehicle. ( i ) HAoECs pre-treated with or without MG132 (500 nM) for 30 min were incubated with 10 –7 M GIP_HUMAN[22–51] and MG132 (100 nM) for 24 h and MMP8 and β-actin mRNA levels were quantified. ** p < 0.01, *** p < 0.001, compared with vehicle. The relative mRNA levels are shown as fold changes (mean ± S.E.M; n = 5).

Article Snippet: For the comparison of the exact expression sites of GIP_HUMAN[22–51] and the mature GIP using dual immunofluorescence staining, we followed the same procedures as described for normal immunohistochemical staining with tissue array, MNO341, except that we used two primary antibodies, the rabbit anti- GIP_HUMAN[22–51] IgG (1:1000) and the mouse anti-human GIP (1:500, R&D Systems) simultaneously, and two secondary antibodies, the goat anti-rabbit-Alexa Fluor 488-conjugated antibody (1:500, Abcam) and the goat anti-mouse-Alexa Fluor 594-conjugated antibody (1:1000, Abcam).

Techniques: Confocal Laser Scanning Microscopy, Cell Culture, Derivative Assay, Fluorescence, Translocation Assay, Incubation, Staining, Western Blot, Expressing

Journal: Scientific Reports

Article Title: GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

doi: 10.1038/s41598-021-93862-w

Figure Lengend Snippet: Proatherosclerotic effects of GIP_HUMAN[22–51] in ApoE −/− mice. The 17-week-old ApoE −/− mice were infused with saline alone ( a , e, and i , vehicle), GIP_HUMAN[22–51] ( b , c , f , g , j, and k , 0.6 nM/kg/h), and/or anti-GIP_HUMAN[22–51] IgG ( c , d , g , h , k, and l , 1.4 μg/kg/h) by osmotic mini-pumps for 4 weeks. The aortic surface was stained with oil red O ( a – d ). Cross sections of the aortic root were stained with oil red O ( e – h ) or anti-MOMA-2 antibody ( i – l ) and counterstained with hematoxylin. Surface area of the atherosclerotic lesions ( m ), cross sectional area of the atherosclerotic lesion ( n ), and macrophage accumulation ( o ) are expressed as means ± SEM ( n = 5–7). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with vehicle.

Article Snippet: For the comparison of the exact expression sites of GIP_HUMAN[22–51] and the mature GIP using dual immunofluorescence staining, we followed the same procedures as described for normal immunohistochemical staining with tissue array, MNO341, except that we used two primary antibodies, the rabbit anti- GIP_HUMAN[22–51] IgG (1:1000) and the mouse anti-human GIP (1:500, R&D Systems) simultaneously, and two secondary antibodies, the goat anti-rabbit-Alexa Fluor 488-conjugated antibody (1:500, Abcam) and the goat anti-mouse-Alexa Fluor 594-conjugated antibody (1:1000, Abcam).

Techniques: Saline, Staining

Journal: Scientific Reports

Article Title: GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

doi: 10.1038/s41598-021-93862-w

Figure Lengend Snippet: Cytokine array screening of mice serum infused with GIP_HUMAN[22–51] for 4 weeks. ( a ) Layout of antibody arrays and abbreviated names of the 111 cytokine/chemokine probes, adapted from the manufacturer’s information. ( b – e ) Complete hybridisation signals after probing with serum samples obtained from ApoE−/− mice infused for 4 weeks with saline alone ( b ), GIP_HUMAN[22–51] ( c ), combined infusion with GIP_HUMAN[22–51] and anti-GIP_HUMAN[22–51] IgG ( d ), or anti-GIP_HUMAN[22–51] IgG ( e ). ( f , g ) Quantified signal intensities of the respective serum proteins in mice infused with GIP_HUMAN[22–51] ( f ) or anti-GIP_HUMAN[22–51] IgG ( g ) in terms of 2-spot mean values relative to untreated experiments are shown.

Article Snippet: For the comparison of the exact expression sites of GIP_HUMAN[22–51] and the mature GIP using dual immunofluorescence staining, we followed the same procedures as described for normal immunohistochemical staining with tissue array, MNO341, except that we used two primary antibodies, the rabbit anti- GIP_HUMAN[22–51] IgG (1:1000) and the mouse anti-human GIP (1:500, R&D Systems) simultaneously, and two secondary antibodies, the goat anti-rabbit-Alexa Fluor 488-conjugated antibody (1:500, Abcam) and the goat anti-mouse-Alexa Fluor 594-conjugated antibody (1:1000, Abcam).

Techniques: Hybridization, Saline

Journal: Scientific Reports

Article Title: GIP_HUMAN[22–51] is a new proatherogenic peptide identified by native plasma peptidomics

doi: 10.1038/s41598-021-93862-w

Figure Lengend Snippet: Systemic expression of GIP_HUMAN[22–51] and its negative colocalisation with GIP in major human organs. ( a–l , a ’– l ’) Human tissue array sections were immunohistochemically stained with either IgG purified from preimmune rabbit serum ( a–l ) or anti-GIP_HUMAN[22–51] IgG at 1:500 dilution ( a ’– l ’). Arrows indicate the positions of positive immunoreactivity. ( a” – l” ) Confocal microscopic images of immunofluorescent dual staining of GIP_HUMAN[22–51] and GIP. Human tissue array sections were double-stained with either the rabbit polyclonal anti-GIP_HUMAN[22–51] antibody (× 1000) or the mouse monoclonal anti-human GIP antibody (× 500). The red signals corresponding to the localisation of GIP were obtained with the Alexa Fluor 594 secondary antibody (× 1000) and the green signals representing GIP_HUMAN[22–51] were obtained with the Alexa Fluor 488 secondary antibody (× 500). The nuclei were counterstained with DAPI (blue). Overlay resulted in yellow signals indicative of colocalisation. ( a–a” ) cerebral cortex, ( b – b ”) cerebellum, ( c – c ”) tonsil, ( d – d ”) lymph node, ( e – e ”) heart, ( f – f ”) liver, ( g – g ”) stomach, ( h – h ”) kidney (glomerulus), ( i – i ”) kidney (tubule), ( j – j ”) small intestine, ( k – k ”) colon and ( l – l ”) testis (scale bar: 100 μm).

Article Snippet: For the comparison of the exact expression sites of GIP_HUMAN[22–51] and the mature GIP using dual immunofluorescence staining, we followed the same procedures as described for normal immunohistochemical staining with tissue array, MNO341, except that we used two primary antibodies, the rabbit anti- GIP_HUMAN[22–51] IgG (1:1000) and the mouse anti-human GIP (1:500, R&D Systems) simultaneously, and two secondary antibodies, the goat anti-rabbit-Alexa Fluor 488-conjugated antibody (1:500, Abcam) and the goat anti-mouse-Alexa Fluor 594-conjugated antibody (1:1000, Abcam).

Techniques: Expressing, Staining, Purification

Journal: JCI Insight

Article Title: ANGPTL8 links refeeding to monocyte dynamics and metabolic inflammation via the CCL5-CCR5 axis

doi: 10.1172/jci.insight.196605

Figure Lengend Snippet: ( A ) Representative images and quantification of monocyte migration induced by conditioned medium (CM) from BMDMs pretreated with or without rA8 over time ( n = 4). ( B ) Heatmap of chemokine-related DEGs (adjusted P < 0.05) in BMDMs cultured with or without rA8 (40 nM) stimulation for 24 hours ( n = 3). ( C ) mRNA expression of chemokines and their receptors after BMDMs were treated with rA8 or scrambled protein ( n = 6 cells examined over 3 independent experiments). ( D ) CCL5 levels in CM from BMDMs with rA8 stimulation over time ( n = 4). ( E ) Migration of BMDMs with or without CCR5 antagonist (TAK-220) before rA8 stimulation ( n = 5 cells examined over 3 independent experiments). ( F ) Effects of chemokine antagonists on migration of BMDMs with rA8 treatment ( n = 4). ( G ) Experimental scheme for CCR5 antagonist (TAK-220) gavage and rA8 injection in Loxp and Angptl8 HepOE mice. ( H ) Plasma proinflammation cytokine levels and ( I ) circulating monocyte populations of indicated groups ( n = 8 mice/group). ( J ) CCL5 levels in CM from human MDMs with or without rA8 stimulation ( n = 4). ( K ) Migration of U937 and THP-1 cells pretreated with or without CCR5 antagonist (TAK-220) before rA8 stimulation ( n = 5 cells examined over 3 independent experiments). Migrated cells were fixed and stained with 0.1% crystal violet ( A and F ). The data are shown as the mean ± SEM and were statistically analyzed by 2-tailed Student’s t test ( A , C , D , F , and J ) or 1-way ANOVA with Tukey’s multiple-comparison test ( E , H , I , and K ). All samples are biologically independent replicates, and n indicates the number of biologically independent samples examined. All the P values were 2 sided, and adjustments were made for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. DEGs, differentially expressed genes; MDMs, monocyte-derived macrophages; rA8, recombinant ANGPTL8; MCP1, monocyte chemoattractant protein-1 (CCL2); MIPs, macrophage inflammatory proteins, RANTES, regulated on activation normal T cell expressed and secreted (CCL5); CM, conditioned medium; TAK-220, CCR5 antagonist; R243, CCR8 antagonist; AMR69, CCL12 inhibitor; SB225002, CXCR2 antagonist; AMG487, CXCR3 antagonist; MIG-2F5.5, anti-CXCL9 antibody; SLW131, CCR7 antagonist.

Article Snippet: The CCR7 antagonist SLW131 (10 nM, MedChem Express, catalog HY-173052), CCR8 antagonist R243 (10 nM, MedChem Express, catalog HY-122219), CCL12 inhibitor AMR69 (10 nM, MedChem Express, catalog HY-B0673), CXCR2 antagonist SB225002 (10 nM, MedChem Express, catalog HY-16711), CXCR3 antagonist AMG487 (10 nM, MedChem Express, catalog HY-15319), and anti-CXCL9 antibody MIG-2F5.5 (10 nM, MedChem Express, catalog HY-P990255) were dissolved and diluted according to the manufacturer’s instructions and were added to medium to inhibit migration.

Techniques: Migration, Cell Culture, Expressing, Injection, Clinical Proteomics, Staining, Comparison, Derivative Assay, Recombinant, Activation Assay