Journal: iScience

Article Title: Uhrf1 governs the proliferation and differentiation of muscle satellite cells

doi: 10.1016/j.isci.2022.103928

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-Myh3 , DSHB , cat# F1.652; RRID: AB_528358.

Techniques: Recombinant, Western Blot, Imaging, Multiplex Assay, Library Quantification, Methylation, Electrophoresis, Software

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A) Transcriptome analysis of indicated genes in testes of ZIKV-infected AG6 mice at 5 dpi. Control mice were injected with PBS. (n = 3 mice for each group). (B and C) Dynamic changes of S100a4 gene level and S100A4+ cells in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at different time points as indicated and subjected to RT-qPCR or immunofluorescence staining (IFA) with anti-S100A4 antibody. (B) Change of S100a4 gene level was expressed as relative expression to β-actin, and shown as means ± SEM. (n = 3–4 mice for each time point). (C) The number of S100A4+ cells were quantified by method described in the experimental procedures, and the number of S100A4+ cells were recorded as cells/mm 2 and was shown as means ± SEM. (n = 5 mice for each time point). (D) IFA for S100A4+ cells. Testes from ZIKV-infected A6 mice were collected at 14 dpi and PBS-injected A6 mice served as controls. S100A4+ cells were detected by anti-S100A4 antibody and nuclei were stained with DAPI. Scale bar, 25 μm. (E-G) Flow cytometry assay for S100A4+ cells. Testicular cells from ZIKV-infected (14 dpi) or PBS-injected A6 mice were subjected to flow cytometry analysis with anti-S100A4 antibody and anti-CD11b antibody. (E) A representative result. (F) Percentage of CD11b+ cells in S100A4+ cells. Results were shown as means ± SEM. (n = 4 mice for each group). (G) Proportion of S100A4+ or S100A4- cells in CD11b+ cells. Results were shown as means ± SEM. (n = 4 mice for each group). (H and I) Co-immunofluorescence staining for S100A4+ cells. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescence staining with anti-S100A4 antibody and (H) anti-F4/80 antibody, or (I) anti-CD45 antibody. Nuclei were shown with DAPI. Scale bar, 50 μm. Relative mRNA expression of S100a4 and proportion in S100A4+ cells or CD11b+ cells in ZIKV-infected testes were analyzed using the Student’s t test, number of S100A4+ cells were analyzed using the Mann-Whitney U test. *p < 0.05 versus Ctrl, **p < 0.01 versus Ctrl.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Control, Injection, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Expressing, Flow Cytometry, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A and B) Distribution of S100A4+ macrophages in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to immunohistochemistry (IHC) staining with anti-S100A4 antibody. (A) Representative images at each time point. Scale bar, 50 μm. (B) Dynamic change of S100A4+ macrophages in seminiferous tubules (STs). The number of S100A4+ macrophages in seminiferous tubules were quantified by method described in the experimental procedures and expressed as cells/mm 2 STs, its number was shown as means ± SEM. (n = 3 mice for each time point). (C-E) Susceptibility of S100A4+ macrophages to ZIKV infection in vitro . Peritoneal macrophages were isolated as described in experimental procedures. (C) Peritoneal macrophages isolated from ZIKV-infected A6 mice at 7 dpi were doubly stained with anti-S100A4 and anti-F4/80 antibodies. (D) Peritoneal macrophages isolated from ZIKV-infected A6 mice at 7 dpi were doubly stained with anti-ZIKV and anti-S100A4 antibodies. Scale bar, 40 μm. (E) Peritoneal macrophages isolated from A6 mice treated with pristane were infected with ZIKV (MOI = 10), and then harvested at different time points as indicated and viral loads was determined by RT-qPCR (n = 3). (F) Susceptibility of S100A4+ macrophages to ZIKV infection in vivo . Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescence staining with anti-S100A4 antibody and anti-ZIKV antibody. Nuclei were shown with DAPI. Scale bar, 25 μm.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Isolation, Immunohistochemistry, In Vitro, Staining, Quantitative RT-PCR, In Vivo, Immunofluorescence

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A-C) Distribution and dynamic of CD8+ cells and their relationship with S100A4+ macrophages in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to IHC staining with anti-CD8α antibody, Scale bar, 50 μm (A) , or co-immunofluorescence staining with anti-CD8α and anti-S100A4 antibodies (B) . A typical seminiferous tubule was outlined with dotted line. Scale bar, 25 μm. (C) The number of CD8+ cells and S100A4+ macrophages in seminiferous tubules were quantified as described in experimental procedures, and expressed as cells/mm 2 STs. (n = 3 mice for each time point). (D) The number of intraluminal S100A4+ macrophages expressing caspase-8 or caspase-3 was quantified as described in experimental procedures, and expressed as cells/mm 2 STs. (n = 3 mice for each time point. See for the representative images). (E-H) Expression of GZMB in S100A4+ macrophages (E) or ZIKV-infected cells (G) . Testicular sections from ZIKV-infected A6 mice at 14 dpi were analyzed with co-immunofluorescence staining using anti-GZMB antibody and anti-S100A4 antibody or anti-ZIKV antibody. Scale bar, 25 μm. GZMB positive cell number of (F) S100A4+ macrophages or (H) ZIKV-infected cells were quantified inside and outside the seminiferous tubules, respectively, and expressed as cells/mm 2 . Results were shown as means ± SEM. (n = 3 mice for each group). Number of GZMB+ S100A4+ macrophages or GZMB+ ZIKV+ cells inside or outside of seminiferous tubules in ZIKV-infected testes were analyzed using the Student’s t test. *p < 0.05, **p < 0.01.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Isolation, Immunohistochemistry, Immunofluorescence, Staining, Expressing

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A) Susceptibility of spermatogenic cells to ZIKV infection. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescence staining with anti-DDX4 antibody and anti-ZIKV antibody. Nuclei were shown with DAPI. Scale bar, 25 μm. (B) Serial sections from ZIKV-infected testes at 14 dpi were subjected to IFA with anti-ZIKV (green), anti-DDX4 (red) or anti-S100A4 (indigo blue) antibodies. Nuclei were visualized with DAPI. Scale bar, 25 μm. (C) The numbers of ZIKV-infected testicular cells by type at 0–28 dpi were quantified as mentioned in experimental procedures and expressed as cells /mm 2 . (n = 3mice for each time point. See for the representative images.) (D-I) Ultrastructure morphological changes of seminiferous tubules from ZIKV-infected A6 mice. (D) A seminiferous tubule was attached by peripheral macrophage-like cells. (E) Peripheral macrophage-like cells in high magnification. (F) Macrophage-like cells accumulated in interstitial space. (G) Macrophage-like cells in outer layer lining seminiferous tubules. (H) Intraluminal macrophage-like cells were surrounded by cell debris. (I) Seminiferous tubules from uninfected A6 mice. M: macrophage-like cells, Sp: spermatogenic cells, SC: sperm cells, Se: Sertoli cells.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Infection, Isolation, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A-E) Testes from PBS-injected or ZIKV-infected A6 mice were collected at 7 dpi and distribution of CLDN1 were analyzed with co-immunofluorescence staining. (A) Distribution of CLDN1 in ZIKV-infected testes revealed by IFA using anti-CLDN1 and anti-S100A4 antibodies. Nuclei were stained with DAPI. Scale bar, 25 μm. (B-E) CLDN1 translocated into nuclei in various testicular cells using antibodies as indicated. (B) and (C) showing CLDN1 translocated into nuclei of spermatogenic cells (B) and their percentage in total of spermatogenic cells (Sps) (C). Scale bar, 25 μm. (D) and (E) showing CLDN1 translocated into nuclei of Sertoli cells (D) and their percentage in total of Sertoli cells (Ses) (E). Scale bar, 25 μm. All data were shown as means ± SEM. (n = 3 mice for each group). (F) Distribution of CLDN1 in ZIKV-infected testes from SA6 mice at 7 dpi. Nuclei were stained with DAPI. Scale bar, 25 μm. (G) The percentage of indicated cells with CLDN1 translocated into nuclei was quantified as described in Experimental Procedure and shown as means ± SEM. (n = 3 mice for each group). All small figures listed at the right side of the corresponding image showing area indicated by arrow in high magnification. Scale bar, 5 μm. Percentage of CLDN1 translocated into nuclei of spermatogenic cells and Sertoli cells in ZIKV-infected testes and control mice were analyzed using the Student’s t test. *p < 0.05, **p < 0.01.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Injection, Infection, Immunofluorescence, Staining, Control

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: (A) Dynamic of IFN-γ concentration in ZIKV-infected testes. Testes from ZIKV-infected A6 mice were isolated at indicated time points and concentration of IFN-γ in testes was measured with Bio-Plex multiplex immunoassays (n = 4 mice for each time point). (B) Percentage of iNOS+ S100A4+ macrophages in total S100A4+ macrophages. Testes from ZIKV-infected A6 mice were isolated at indicated time points and subjected to co-immunofluorescent staining with anti-S100A4 and anti-iNOS or anti-CD163 antibodies. See for representative images. The percentage of iNOS+ S100A4+ macrophages was quantified as described in experimental procedure. (n = 3 mice for each time point). (C) Expression of IFN-γ in S100A4+ macrophages. Testis sections from PBS-injected or ZIKV-infected A6 mice at 14 dpi were analyzed using co-immunofluorescent staining with anti-S100A4 and anti-IFN-γ antibodies. Nuclei were stained with DAPI. Scale bar, 25 μm. (D) Effect of IFN-γ on CLDN1 distribution in vitro . Sertoli cells were treated with 50 ng IFN-γ at 32°C and were collected at 0, 24 and 48h. Distribution of CLDN1 was visualized by immunofluorescent staining. Nuclei were stained with DAPI. Scale bar, 25 μm. (E) Effect of IFN-γ on CLDN1 redistribution in vivo . A6 mice were intravenously injected with IFN-γ (4 μg per mouse daily) or PBS for 10 days. At 10 day after treatment, testes were collected and distribution of CLDN1 in testicular cells was analyzed by IFA. Scale bar, 25 μm. (F) Expression of CLDN1 and Occludin in testes from ZIKV-infected A6 and AG6 mice as well as their corresponding controls. Mice were challenged with ZIKV or injected with PBS, and testes were isolated at 7 dpi and subjected to Western Blot. (n = 3 for each group). (G) Distribution of CLDN1 in ZIKV-infected AG6 testes. CLDN1 distribution in testes from PBS-injected or ZIKV-infected AG6 mice (7 dpi) were analyzed using co-immunofluorescent staining. Nuclei were stained with DAPI. Scale bar, 25 μm. (H-J) Susceptibility of testicular cells to ZIKV in A6 or AG6 mice. Co-localization of ZIKV antigens with various cell marker molecules in testes from ZIKV-infected A6 or AG6 mice (7 dpi) was analyzed using co-immunofluorescent staining with anti-ZIKV antibody and (H) anti-DDX4 antibody, (I) anti-SOX9 antibody or (J) anti-α-SMA antibody. Nuclei were stained with DAPI. Scale bar, 50 μm. IFN-γ concentration in ZIKV-infected testis tissues were analyzed using the Student’s t test. *p < 0.05, **p < 0.01.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques: Concentration Assay, Infection, Isolation, Multiplex Assay, Staining, Expressing, Injection, In Vitro, In Vivo, Western Blot, Marker

Journal: PLoS Pathogens

Article Title: S100A4+ macrophages facilitate zika virus invasion and persistence in the seminiferous tubules via interferon-gamma mediation

doi: 10.1371/journal.ppat.1009019

Figure Lengend Snippet: Schematic diagram to show that S100A4+ macrophages assist ZIKV to infect mice testes and persist in seminiferous tubules.

Article Snippet: The testicular cells were then incubated with fluorochrome-conjugated antibodies to CD11b-APC (1:100, e-bioscience, 17-0112-81) or fluorochrome-unconjugated rabbit anti-mouse CD8α antibody (1:100, Cell Signaling Technology, D4W2Z), rabbit anti-mouse CD4 antibody (1:100, Abcam, ab183685), rabbit anti-mouse S100A4 antibody (1:100, Cell Signaling Technology, 13018S), mouse anti-mouse S100A4 antibody (1:100, proteintech, 66489–1), rabbit anti-mouse DDX4 antibody (1:100, Abcam, ab13840), rabbit anti-mouse SOX9 antibody (1:100, Abcam, AB5535) or rabbit anti-mouse α-SMA antibody (1:100, Abcam, ab5694).

Techniques:

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: Mixed neuronal-glial cerebrocortical cultures from WT or IFNAR1KO mice were incubated with IFNβ in increasing doses (from 500 to 5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h. Total RNA was extracted from cell lysates, analyzed by qRT-PCR and normalized to GAPDH expression levels. ( a–d ) RNA expression is shown as fold change (FC) in relation to vehicle treated controls which were defined as baseline activity. ( e–h ) Time course for protein expression measured in cell-free supernatants for CCL3, CCL4, CCL5 and CXCL10 using a commercially available multiplex assay as described in Methods. Baseline protein expression in vehicle treated cell cultures is represented as 0 h time point. Values are mean ± s.e.m.; n = 3–5 independent experiments per ISG; ***p < 0.001, **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test. For clarity, the significance is only indicated for differences between treatments and baseline within each experimental group.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Incubation, Control, Quantitative RT-PCR, Expressing, RNA Expression, Activity Assay, Multiplex Assay

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: ( a ) Mixed neuronal-glial cerebrocortical cultures from WT mice were simultaneously exposed for 3 days to HIV gp120 BaL (200 pM) and mouse IFNβ (5,000 U/ml) in the presence and absence of neutralizing antibodies against CCL3, CCL4, CCL5, IFNγ or CXCL10. IFNγ antibody was used as control for neutralizing antibodies since this protein was undetectable in cerebrocortical cell cultures. ( b ) Mouse cerebrocortical cultures from IFNAR1KO mice were stimulated with gp120 BaL for 24 h the presence or absence of mouse IFNβ (5,000 U/ml) or BSA/PBS control. ( c ) Cerebrocortical cell cultures from WT mice were simultaneously exposed for 3 days to HIV gp120 BaL in the presence and absence of murine CCL4 (2 or 20 nM). Neuronal survival was assessed by immunofluorescence microscopy and counting of MAP-2/NeuN double-positive neurons. Values are mean ± s.e.m.; n = 3–5 independent experiments with 3–7 replicates and an average of 9,000 (IFNAR1KO) or 5,700 (WT) cells counted per condition; ** p < 0.01, *** p < 0.001 by ANOVA with Fisher’s PLSD post hoc test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Control, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: RNA was purified from one brain hemisphere each of 4–5 month-old HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle and analyzed by qRT-PCR for fold-change (FC) in ISG expression. Significant changes in gene expression were observed between IFNβ and vehicle treatment groups in WT brains ( a ) for CCL4, and in gp120tg brains ( b ) for CCL4, CXCL11 and IRF3. Expression of transgenic HIVgp120 was not affected by IFNβ ( c ). Values are mean ± s.e.m.; n = 4–5 animals per group/genotype; *p < 0.05, student’s t-test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Purification, Quantitative RT-PCR, Expressing, Gene Expression, Transgenic Assay

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: Sagittal brains sections of HIVgp120tg and WT littermate mice previously treated with intranasal IFNβ or vehicle (veh) were immunolabeled for CCL4, neuronal MAP-2 or astrocytic GFAP. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was labeled with Hoechst (H) 33342. The fluorescence-labeled brain sections were analyzed using confocal laser-scanning microscopy. Representative images of cortex layer III are shown; scale bar, 50 μm.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Immunolabeling, Labeling, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Scientific Reports

Article Title: IFNβ Protects Neurons from Damage in a Murine Model of HIV-1 Associated Brain Injury

doi: 10.1038/srep46514

Figure Lengend Snippet: ( a ) Cerebrocortical cultures from mice were prepared to either contain microglia, neurons and astrocytes (M + N + A) or were depleted of microglia (N + A) or neurons and microglia (A). Complete and depleted cell cultures were incubated with mIFNβ (5,000 U/ml) or BSA/PBS vehicle control for 0, 3, 6, 12 and 24 h and concentrations of CCL4 were measured in cell-free supernatants using a commercially available multiplex assay as described in Methods. Maximum concentrations were reached in samples of 12 to 24 h mIFNβ exposure and compared to vehicle-treated, baseline samples. Values are mean ± s.e.m.; n = 3 independent experiments; *p < 0.05, student’s t-test. ( b ) Microglia-depleted rat cerebrocortical cultures were exposed for 24 h to 50% cell-free conditioned media (CM) from human MDM in the presence or absence of human IFNβ (5,000 U/ml). MDM were previously stimulated for 24 h with HIV-1 gp120 BaL (MDM gp120 CM) or vehicle (MDM CM). Following the incubation the cells were fixed and permeabilized. Neurons were immunolabeled for neuronal MAP-2 and NeuN and nuclear DNA was stained with H33342. Neuronal survival was assessed using fluorescence microscopy and cell counting as described in Methods. Values are mean ± s.e.m.; n = 2 independent experiments, with 4–8 replicates and an average of 4,000 cells counted per condition; **p < 0.01, *p < 0.05 by ANOVA with Fisher’s PLSD post hoc test.

Article Snippet: For visualization of CCL4, mouse brain sections were immunolabeled with primary antibodies for CCL4 (ProSci, Poway, CA, cat# 7227, 1:50) in combination with Ab against MAP-2 or GFAP for 24 h. Alexa Fluor 488, 555 and 647 conjugated secondary antibodies were employed to visualize primary Abs and nuclear DNA was stained with Hoechst (H) 33342.

Techniques: Incubation, Control, Multiplex Assay, Immunolabeling, Staining, Fluorescence, Microscopy, Cell Counting

Journal: Nature protocols

Article Title: Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

doi: 10.1038/s41596-021-00577-3

Figure Lengend Snippet: a, (Steps 1-7) Production of the substrate begins using a ccDNA with the oligonucleotide-binding sequence of interest. (Steps 8-21) A nick is introduced into the plasmid on the strand that will modified with a site-specific DNA lesion. (Steps 22-29) The nicked strand is digested using Exo III to generate circular ssDNA. (Steps 30-33) An oligonucleotide containing the lesion of interest is annealed to the ssDNA. (Steps 34-42) The annealed plasmid:oligo duplex is incubated with T4 Polymerase and T4 Ligase produce ccDNA containing the lesion of interest. (Steps 42-69) Pure closed circular product is obtained through incubation with T5 Exo followed by Proteinase K digest, DNA extraction and precipitation, and dissolution in TE buffer. Note, procedures in steps 59-68 result in linearized plasmids. All yields refer to reporter plasmids generated using the oligonucleotide extension methodology. (Steps 70-101) substrates are subjected to a battery of in vitro and in vivo QC steps. b, Representative agarose gel electrophoretic analysis of starting ccDNA (Lane 2), ocDNA (Lane 3), circular ssDNA (Lane 4) and the final purified product (Lane 5) are shown. c, Representative gel analysis showing evidence of incomplete digest of ccDNA with nicking enzyme (Lane 3) and incomplete digest of nicked DNA with Exo III (Lane 5). d, Overexposed representative gel analysis showing evidence of residual ssDNA due to incomplete extension when the molar ratio of oligonucleotide to ssDNA is too low (1:1, Lane 5 and 1:2 Lane 6) and an absence of ssDNA when the ratio is optimal (4:1, Lane 7). e, Representative gel analysis showing accumulation of open circular DNA in a preparation where the proteinase K step was excluded (Lane 5), and the near absence of nicked product when proteinase K is included (Lane 6). In panels b-e, the relevant steps of the protocol are indicated below the gel.

Article Snippet: In a 1.8 mL microcentrifuge tube, prepare an annealing reaction between the custom 5’-phosporylated oligo and the ssDNA, using a 4:1 molar ratio of phosphorylated oligo to single stranded DNA with 1X NEBuffer 2.1 as shown in the example below: Example: To anneal phosphorylated oligo to 100 μg (~91 pmol) ssDNA Add 50 μL single stranded DNA (initial concentration 2000 ng/μL; final concentration 833 ng/μL) Add 12 μL 10X NEBuffer 2.1 (1X) Add 3.6 μL 100μM custom oligonucleotide (360 pmol; 16 μM) Add 54 μL DEPC-treated water (up to a total volume of 120 μL) Mix thoroughly by pipetting.

Techniques: Binding Assay, Sequencing, Plasmid Preparation, Modification, Incubation, DNA Extraction, Generated, In Vitro, In Vivo, Agarose Gel Electrophoresis, Purification

Journal: Nature protocols

Article Title: Large-scale preparation of fluorescence multiplex host cell reactivation (FM-HCR) reporters

doi: 10.1038/s41596-021-00577-3

Figure Lengend Snippet: Troubleshooting tips for potential problems.

Article Snippet: In a 1.8 mL microcentrifuge tube, prepare an annealing reaction between the custom 5’-phosporylated oligo and the ssDNA, using a 4:1 molar ratio of phosphorylated oligo to single stranded DNA with 1X NEBuffer 2.1 as shown in the example below: Example: To anneal phosphorylated oligo to 100 μg (~91 pmol) ssDNA Add 50 μL single stranded DNA (initial concentration 2000 ng/μL; final concentration 833 ng/μL) Add 12 μL 10X NEBuffer 2.1 (1X) Add 3.6 μL 100μM custom oligonucleotide (360 pmol; 16 μM) Add 54 μL DEPC-treated water (up to a total volume of 120 μL) Mix thoroughly by pipetting.

Techniques: Transformation Assay, Plasmid Preparation, Selection, Agarose Gel Electrophoresis, Incubation, Sequencing, Mutagenesis, Activity Assay, Spectrophotometry, Transferring, Concentration Assay, Ethanol Precipitation, Modification, Ligation, Purification, Nucleic Acid Electrophoresis, In Vitro, Flow Cytometry, Software, Fluorescence, Multiplexing, Quantitation Assay, Expressing, Transfection, IF-cells, Staining

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) Metabolic gene set analysis of RNAseq data provided by the TCGA, classified according to KEGG (Li et al., 2014). Generated metabolic gene sets were ranked based on their median fold expression changes in ccRCC tumor (n=480) vs. normal tissue (n=69), and plotted as median ± median absolute deviation. (B) The complete urea cycle as configured in the liver. Inset: TCGA-derived gene expression changes of urea cycle enzymes in ccRCC. (C) Copy number variation and mutational burden of urea cycle enzymes in 184 ccRCC tumors (data from TCGA). (D) ARG2 and ASS1 mRNA levels in ccRCC (TCGA). ***p <0.001, Welch’s t-test. (E) Copy number variation in ARG2 and ASS1 in ccRCC patients. Kaplan-Meier survival analysis of copy number loss in ARG2 and ASS1 (from TCGA data). Mantel-Cox log-rank test was performed. (F) Representative immunohistochemistry images of ARG2 or ASS1 protein in primary ccRCC, N = normal, T = tumor. Scale bars represent 100 μm (G) Violin plot of urea cycle metabolite abundance in primary ccRCC combining two independent datasets, n = 158 (Hakimi et al., 2016; Li et al., 2014). Data are displayed as the log2 tumor/normal fold change and are pseudo-colored according to the intensity of the fold change in median. The internal bars represent the mean and SD of 158 tumor/normal pairs. Abbreviations: ARG2, arginase 2; ASS1, argininosuccinate synthase 1; ORNT1, ornithine translocase 1 (also called SLC25A15); CPS1, carbamoylphosphate synthase 1; OTC, ornithine transcarbamylase. See also Figure S1 and Table S1.

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Generated, Expressing, Derivative Assay, Gene Expression, Immunohistochemistry

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) 786-O xenograft tumor growth in nude mice, where cells re-express ASS1, wild type ARG2, ARG2H160F mutant, or both ASS1 and ARG2 (n = 10 mice in each cohort, except for n=5 for the ARG2 H160F study. (B) Immunohistochemistry staining for Ki67 as a marker of proliferation in xenograft tumors expressing urea cycle enzymes, high power fields (HPF). (C) mTORC1 activity in ARG2- or ASS1- expressing xenograft tumors, as assessed by the mTORC1 targets phospho-S6K1, phospho-S6, and phospho-4E-BP1. (D) Unbiased metabolomic analysis of control or ARG2 expressing xenograft tumors, n = 7 for each cohort. ARG2 expression causes robust global reductions in several categories of metabolites, especially lipids, nucleotides, and biosynthetic co-factors. (E) Amino acid abundance in 786- O xenograft tumors (n = 7). Data are represented as the percent change in amino acid, after normalization to each corresponding control tumor. *p < 0.05, **p < 0.01, ***p < 0.001, Welch’s t-test was used for each dataset. See also Figures S3 and S4, and Table S2.

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Mutagenesis, Immunohistochemistry, Staining, Marker, Expressing, Activity Assay, Control

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) ARG2 and ASS1 protein levels in HK-2 kidney epithelial and various ccRCC cell lines. Tubulin or actin served as loading controls. (B) Upper panel: short hairpin-mediated knockdown of ARG2, ASS1, or both simultaneously (Double KD) in HK-2 cells. Tubulin or actin served as loading controls. Lower panel: mRNA levels after knockdown with two independent hairpins. Error bars represent SEM of 3 independent replicates. (C-D) Knockdown of ARG2 or ASS1 augments HK-2 cell growth in (C) 2D growth and (D) 3D soft agar colony formation assays. Double KD experiments employed ARG2 SH-1 and ASS1 SH-4. In (C), cells were grown in 1% FBS and 1 mM glucose media (changed every 2 days). Error bars represent SEM from 3 replicate wells. **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. (E) Clonally selected CRISPR/Cas9-mediated ARG2−/− or ASS1−/− HK-2 cells demonstrate enhanced growth. Guide RNA against EGFP was used as a control, ns = non-specific band. Cells were grown in 1% FBS and 1 mM glucose media (changed every 2 days). Error bars represent SEM of 16 replicate wells. See also Figure S2.

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Knockdown, CRISPR, Control

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) Left panel: Ectopic expression of ARG2 and/or ASS1 in 786-O ccRCC polyclonal populations. Numbers below ARG2 and ASS1 western images represent the quantified fold change in protein compared to normal HK-2 cells. Right panel: 3D spheroid growth assay of 786-O cells. After three weeks of growth, spheroid volume was measured via microscopy and the number of cells per spheroid was counted after accutase digestion of spheroids to a single cell suspension. Error bars represent SEM of three independent replicates containing 12 spheroids each. B) Soft agar growth assays of 786-O ccRCC cells. Error bars represent SEM of 5 replicate wells. (C) LC/MS quantitation of arginine, citrulline, and aspartate in 786-O cells stably expressing urea cycle enzymes. Error bars represent SEM of 4 replicates. (D) Expression and activity of wild type (WT) and H160F mutant ARG2 and (E) 786-O spheroid growth assay demonstrating ARG2 catalytic activity-dependent growth suppression. Error bars represent SEM of 3 independent replicates containing 12 spheroids each. (F) 786-O soft agar colony growth assay depicting ARG2 catalytic activity-dependent growth suppression. Error bars represent SEM of 3 replicate wells. *p < 0.05, **p < 0.01, ***p < 0.001, a one-way ANOVA with Tukey’s multiple comparisons test was conducted for each dataset unless otherwise specified.

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Expressing, Western Blot, Growth Assay, Microscopy, Suspension, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Stable Transfection, Activity Assay, Mutagenesis

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) Schematic representation of 15N4-Arginine labeling into glutamate and proline synthesis. Ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) reactions and their requirement for pyridoxal phosphate (PLP) as a cofactor are highlighted. Closed red circles represent heavy nitrogen (15N) while open black circles represent carbon atoms (12C). (B) ARG2 controls the downstream synthesis of glutamate and proline. HK-2 (left panel) or 786-O cells (right panel) incubated with 15N4-Arginine (same conditions as in Figure 6B). Synthesis of glutamate and proline from arginine results in one -15N incorporation (M1 enrichment), represented as atom percent excess (APE). Error bars represent SEM of 3 and 5 replicates for HK-2 and 786-O, respectively. (C) Schematic for the synthesis of PLP. Several precursor forms of pyridoxal exist and are interconverted between their phosphorylated versions; only PLP is the bioactive cofactor. (D) mRNA levels of pyridoxal kinase (PDXK) in ccRCC (data from TCGA). Box plots represent 69 normal and 480 tumor samples, one-way ANOVA with Tukey’s multiple comparisons test. (E) ARG2 directly modulates the levels of bioactive PLP. HK-2 (left panel) or 786-O cells (right panel) were grown in 1% FBS with 1 mM glucose for 24 hours (HK-2) or hypoxia (0.5% O2) for 48 hours (786-O) prior to quantifying the total PL, PLP, PN, and PM levels via LC/MS. Error bars represent SEM of 3 and 5 replicates for HK-2 and 786-O, respectively. (F) Ectopic ARG2 and/or PDXKexpression in 786-O cells. (G) PDXK overexpression alters levels of pyridoxine pathway components. 786-O cells were cultured under hypoxia (0.5% O2) for 48 hours prior to quantifying total PL, PLP, PN, and PM levels via LC/MS. Error bars represent SEM of 5 replicates. (H-I) PDXK expression reverses ARG2-dependent growth suppression. 3D soft agar colony formation assay of 786-O cells ectopically expressing either ARG2 and/or PDXK. (H) Quantification of colony number and (I) representative pictures of colony size. Scale bar represents 25 μm. Error bars represent SEM of 6 replicate wells for each condition, one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, a two-way ANOVA with Tukey’s multiple comparisons test was conducted for each dataset unless otherwise specified. See also Figures S5 and S6.

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Labeling, Incubation, Liquid Chromatography with Mass Spectroscopy, Over Expression, Cell Culture, Expressing, Soft Agar Assay

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: (A) Schematic representation of 15N4-arginine contribution to polyamine biosynthesis. Closed red circles represent heavy nitrogen (15N) while open black circles represent carbon atoms (12C). (B) ARG2 increases arginine utilization for polyamines. Top panel, HK-2 cells grown in 1% FBS and 1 mM glucose for 24 hours and then supplemented with 15N4-arginine for an additional 24 hours. M2 enrichment represents the proportion of each metabolite containing two - 15N atoms (atom percent excess [APE]). Bottom panel, 786-O cells grown under hypoxia (0.5% O2) for 48 hours and then supplemented with 15N4 Arginine for an additional 24 hours. Error bars represent SEM of 5 replicates. (C) 786-O cells grown under normoxia or hypoxia (0.5% O2) for 48 hours followed by quantification of total polyamine pools with LC/MS. Error bars represent SEM of 8 replicates. (D-E) HK-2 cells grown in 1% FBS and 1 mM glucose and supplemented with various concentrations of putrescine or spermine. Error bars represent SEM of 8 replicates. (F) CRISPR/Cas9 mediated deletion of ornithine decarboxylase (ODC1) in ARG2 reconstituted 786-O cells. Numbers below ODC1 western blot images represent the quantified fold change in protein compared to 786-O lines with vector or ARG2, respectively. (G) ODC1 loss in 786-O cells reverses ARG2-mediated growth suppression in a soft agar colony formation assay. Error bars represent SEM of 3 replicate wells. *p < 0.05, **p < 0.01, ***p < 0.001, a two-way ANOVA with Tukey’s multiple comparisons test was conducted for each dataset unless otherwise specified. See also Figure S7.

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Liquid Chromatography with Mass Spectroscopy, CRISPR, Western Blot, Plasmid Preparation, Soft Agar Assay

Journal: Cell metabolism

Article Title: Arginase 2 Suppresses Renal Carcinoma Progression via Biosynthetic Cofactor Pyridoxal Phosphate Depletion and Increased Polyamine Toxicity

doi: 10.1016/j.cmet.2018.04.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Predesigned Taqman primers were obtained from Life Technologies for the following genes: ACTB (HS01060665_G1), 18S (HS03928985_G1), ASS1 (HS01597989_G1), and ARG2 (HS00982833_M1).

Techniques: Recombinant, Multiplex sample analysis, Arginase Activity Assay, Colorimetric Assay, Mutagenesis, Software

Journal: eLife

Article Title: CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions

doi: 10.7554/eLife.60646

Figure Lengend Snippet: ( A ) Peritoneal macrophages were isolated, polarized to M1, and then treated with 500 μg/ml CXCR3 NAB or isotype control IgG. After 48 hr treatment, samples were analyzed for expression of iNOS or Ym1 using immunofluorescence. Images shown are representative of three independent experiments done on peritoneal macrophages obtained from three mice (biological replicates). The scale bar indicates 100 μm. ( B ) Peritoneal macrophages were isolated, polarized to M1, and treated with 500 μg/ml CXCR3 NAB or isotype control IgG. After 48 hr, samples were analyzed by qPCR for expression of M1 macrophage marker Cd38 and M2 macrophage markers Ym1/ Chil3 , Arg1 , and Fizz1/ Retnla . Results shown are representative of three independent experiments done on peritoneal macrophages obtained from three mice (biological replicates). Statistical analysis using the Student’s t-test indicates significance (marked by an asterisk, Cd38 : p-value=0.0009, Ym1/ Chil3 : p-value<0.0001, Arg1 : p-value<0.0001, Fizz1/ Retnla : p-value<0.0001). Error bars indicate standard deviation. ( C ) Pancreatic abnormal areas from KC mice treated with CXCR3 NAB or isotype control IgG were analyzed for presence of inflammatory macrophages (co-immunofluorescence for F4/80 and pY701-STAT1). Shown is a representative area from staining and analysis done on three mice per group. The H and E staining highlights the region analyzed. The scale bar indicates 50 μm. ( D ) Quantification of pY701-STAT1 + macrophages in pancreata from KC mice (n = 3 mice per treatment group) treated with CXCR3 NAB or isotype control IgG. Cells were counted in three representative fields per mouse. The arcsin transformation was done on the proportion of macrophages which were pY701-STAT1 + . Statistical analysis using the Student’s t-test indicates significance between biological replicates (indicated by an asterisk, p-value=0.0004). Error bars indicate standard deviation. Figure 3—source data 1. qPCR for Cd38, Ym1/ Chil3 , Arg1 , and Fizz1 /Retnla in M1 polarized macrophages treated with CXCR3 NAB or isotype control IgG, and quantification of pY701-STAT1+ macrophages in KC mice treated with CXCR3 NAB or isotype control IgG (panels B and D).

Article Snippet: Sequence-based reagent , Arg1 , TaqMan (Thermo Fisher Scientific) , Mm00475988_m1 , qPCR probe.

Techniques: Isolation, Control, Expressing, Immunofluorescence, Marker, Standard Deviation, Staining, Transformation Assay

Journal: eLife

Article Title: CXCL10/CXCR3 signaling contributes to an inflammatory microenvironment and its blockade enhances progression of murine pancreatic precancerous lesions

doi: 10.7554/eLife.60646

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , Arg1 , TaqMan (Thermo Fisher Scientific) , Mm00475988_m1 , qPCR probe.

Techniques: Control, RNAscope, In Situ Hybridization, Multiplex Assay, Recombinant, Sequencing, Software, Flow Cytometry, Cell Culture, Plasmid Preparation