Structured Review

The Jackson Laboratory wt c57bl 6 mice
Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into <t>C57BL/6</t> mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P
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1) Product Images from "Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)"

Article Title: Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI127755

Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P
Figure Legend Snippet: Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Clone Assay, Transduction, Plasmid Preparation, Over Expression, Staining, Flow Cytometry, Mouse Assay

Tumor cell–intrinsic Epha2 regulates sensitivity to immunotherapy. ( A and B ) Epha2 -WT and Epha2 -KO tumor growth and mouse survival with or without the GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 4–8/group). GAFCP treatment started 12 days after implantation, at 3–5 mm tumor diameter. ( C ) Size change of Epha2 -WT and Epha2- KO tumors from indicated clones relative to the baseline after 3 weeks with or without GAFCP treatment. PDA tumor cells were implanted subcutaneously into C57BL/6 mice ( n = 4–8/group) and treated with GAFCP for 12 days (average tumor diameter 3–5 mm at the treatment start). Statistical differences between groups calculated by linear mixed-effects model with Tukey’s HSD post test using the lme4 in R ( A and B , left). The log-rank P values for Kaplan-Meier survival curves were calculated in GraphPad Prism ( A and B , right). *** P
Figure Legend Snippet: Tumor cell–intrinsic Epha2 regulates sensitivity to immunotherapy. ( A and B ) Epha2 -WT and Epha2 -KO tumor growth and mouse survival with or without the GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 4–8/group). GAFCP treatment started 12 days after implantation, at 3–5 mm tumor diameter. ( C ) Size change of Epha2 -WT and Epha2- KO tumors from indicated clones relative to the baseline after 3 weeks with or without GAFCP treatment. PDA tumor cells were implanted subcutaneously into C57BL/6 mice ( n = 4–8/group) and treated with GAFCP for 12 days (average tumor diameter 3–5 mm at the treatment start). Statistical differences between groups calculated by linear mixed-effects model with Tukey’s HSD post test using the lme4 in R ( A and B , left). The log-rank P values for Kaplan-Meier survival curves were calculated in GraphPad Prism ( A and B , right). *** P

Techniques Used: Mouse Assay, Clone Assay

2) Product Images from "Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)"

Article Title: Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI127755

Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P
Figure Legend Snippet: Tumor cell–intrinsic Ptgs2 promotes T cell–low TME and resistance to immunotherapy in PDA. ( A ) Relative expression of Ptgs2 mRNA measured by qPCR in T cell–high PDA tumor cell clones transduced with either empty vector (EV) or pCDH-FHC vector carrying mouse Ptgs2 gene for overexpression ( Ptgs2 -OE). Representative data from n = 3 independent experiments. ( B and C ) Representative immunofluorescent staining images ( B ) and quantification ( C ) of CD3 + T cells in 2838c3-EV and 2838c3- Ptgs2 -OE subcutaneous tumors ( n = 5/group): CD3 (red), YFP (green), and DAPI (blue). Original magnification, ×20. ( D – F ) Flow cytometric analysis of subcutaneously implanted empty vector or Ptgs2 -OE tumors from indicated clones ( n = 5-7/group). ( G ) Parental and Ptgs2 -OE T cell–high tumor growth and mouse survival with or without GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 5–8/group). GAFCP treatment started 9 days after implantation at 3–5 mm tumor diameter. ( A , C – F ) Data are presented as boxplots, with horizontal lines and error bars indicating mean and range, respectively. Statistical analysis between 2 groups calculated using Student’s unpaired t test ( C – F ). The log-rank P values for Kaplan-Meier curves in G were calculated using GraphPad Prism. Statistical analysis of tumor growth curves performed using linear mixed-effects model with Tukey’s HSD post test using lme4 and the survival package in R ( G ). * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Clone Assay, Transduction, Plasmid Preparation, Over Expression, Staining, Flow Cytometry, Mouse Assay

Tumor cell–intrinsic Epha2 regulates sensitivity to immunotherapy. ( A and B ) Epha2 -WT and Epha2 -KO tumor growth and mouse survival with or without the GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 4–8/group). GAFCP treatment started 12 days after implantation, at 3–5 mm tumor diameter. ( C ) Size change of Epha2 -WT and Epha2- KO tumors from indicated clones relative to the baseline after 3 weeks with or without GAFCP treatment. PDA tumor cells were implanted subcutaneously into C57BL/6 mice ( n = 4–8/group) and treated with GAFCP for 12 days (average tumor diameter 3–5 mm at the treatment start). Statistical differences between groups calculated by linear mixed-effects model with Tukey’s HSD post test using the lme4 in R ( A and B , left). The log-rank P values for Kaplan-Meier survival curves were calculated in GraphPad Prism ( A and B , right). *** P
Figure Legend Snippet: Tumor cell–intrinsic Epha2 regulates sensitivity to immunotherapy. ( A and B ) Epha2 -WT and Epha2 -KO tumor growth and mouse survival with or without the GAFCP treatment. Tumor cells implanted subcutaneously into C57BL/6 mice ( n = 4–8/group). GAFCP treatment started 12 days after implantation, at 3–5 mm tumor diameter. ( C ) Size change of Epha2 -WT and Epha2- KO tumors from indicated clones relative to the baseline after 3 weeks with or without GAFCP treatment. PDA tumor cells were implanted subcutaneously into C57BL/6 mice ( n = 4–8/group) and treated with GAFCP for 12 days (average tumor diameter 3–5 mm at the treatment start). Statistical differences between groups calculated by linear mixed-effects model with Tukey’s HSD post test using the lme4 in R ( A and B , left). The log-rank P values for Kaplan-Meier survival curves were calculated in GraphPad Prism ( A and B , right). *** P

Techniques Used: Mouse Assay, Clone Assay

3) Product Images from "Characterization of a Novel Bispecific Antibody That Activates T Cells In Vitro and Slows Tumor Growth In Vivo"

Article Title: Characterization of a Novel Bispecific Antibody That Activates T Cells In Vitro and Slows Tumor Growth In Vivo

Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

doi: 10.1089/mab.2019.0035

CD3xEpCAM treatment increased survival and reduced tumor growth of B16F10/EpCAM-treated mice. Sixteen C57BL/6 mice were implanted with B16F10/EpCAM tumors. Eight mice were treated biweekly with 60 μg of CD3xEpCAM antibody per dose (green line) or 60 μg of CD3xnull antibody over the course of 2 weeks. Blue arrows indicate antibody injections. (A) Average tumor growth of CD3xEpCAM- and CD3xnull-treated mice. Error bars are standard error of the mean. (B) Survival plot of CD3xEpCAM- and CD3xnull-treated groups. (C) Spider plots for CD3xnull (purple lines) or CD3xEpCAM (green lines)-treated mice. ** p
Figure Legend Snippet: CD3xEpCAM treatment increased survival and reduced tumor growth of B16F10/EpCAM-treated mice. Sixteen C57BL/6 mice were implanted with B16F10/EpCAM tumors. Eight mice were treated biweekly with 60 μg of CD3xEpCAM antibody per dose (green line) or 60 μg of CD3xnull antibody over the course of 2 weeks. Blue arrows indicate antibody injections. (A) Average tumor growth of CD3xEpCAM- and CD3xnull-treated mice. Error bars are standard error of the mean. (B) Survival plot of CD3xEpCAM- and CD3xnull-treated groups. (C) Spider plots for CD3xnull (purple lines) or CD3xEpCAM (green lines)-treated mice. ** p

Techniques Used: Mouse Assay

CD3xEpCAM murine bispecific antibody binds mouse CD3 and human EpCAM and redirected OT1 T cells to kill human EpCAM expressing B16F10. (A) CD3xEpCAM mouse bispecific antibody had anti-murine CD3ɛ arm (clone 2C11) and anti-human EpCAM arm (clones 4–7). ( 15 ) (B) B16F10/EpCAM cells were incubated with 10 μg/mL of either CD3xEpCAM or CD3xnull and EpCAMxnull controls. Antibody binding to human EpCAM on B16F10 was detected using anti-mouse IgG APC secondary antibody. Secondary antibody stained cells only (gray-filled histograms) were used as a negative control, and open histograms denotes staining with CD3xEpCAM bispecific antibody of either B16F10/EpCAM (B) or OT1 T cells (C) . (D, E) The CD3xEpCAM antibody redirected OT1 T cells isolated from spleen and lymph nodes of OT1 mice and pre-activated with SIINFEKL peptide loaded C57BL/6 mature DC for 48–72 hours as demonstrated by BADTA/Eu-based CTL. (D) E:T ratio was varied in the presence of 10 μg/mL of either CD3xEpCAM (red circles) or CD3xnull negative control (blue squares). (E) The concentration of either CD3xEpCAM (red circles) or CD3xnull (blue circles) was varied from 0.006 to 5 μg/mL, and the E:T is kept constant at 10:1. The data are representative of three individual experiments. APC, allophycocyanin; CTL, cytotoxic T lymphocytes; DC, dendritic cell; EpCAM, epithelial cell adhesion molecule; IgG, immunoglobulin G.
Figure Legend Snippet: CD3xEpCAM murine bispecific antibody binds mouse CD3 and human EpCAM and redirected OT1 T cells to kill human EpCAM expressing B16F10. (A) CD3xEpCAM mouse bispecific antibody had anti-murine CD3ɛ arm (clone 2C11) and anti-human EpCAM arm (clones 4–7). ( 15 ) (B) B16F10/EpCAM cells were incubated with 10 μg/mL of either CD3xEpCAM or CD3xnull and EpCAMxnull controls. Antibody binding to human EpCAM on B16F10 was detected using anti-mouse IgG APC secondary antibody. Secondary antibody stained cells only (gray-filled histograms) were used as a negative control, and open histograms denotes staining with CD3xEpCAM bispecific antibody of either B16F10/EpCAM (B) or OT1 T cells (C) . (D, E) The CD3xEpCAM antibody redirected OT1 T cells isolated from spleen and lymph nodes of OT1 mice and pre-activated with SIINFEKL peptide loaded C57BL/6 mature DC for 48–72 hours as demonstrated by BADTA/Eu-based CTL. (D) E:T ratio was varied in the presence of 10 μg/mL of either CD3xEpCAM (red circles) or CD3xnull negative control (blue squares). (E) The concentration of either CD3xEpCAM (red circles) or CD3xnull (blue circles) was varied from 0.006 to 5 μg/mL, and the E:T is kept constant at 10:1. The data are representative of three individual experiments. APC, allophycocyanin; CTL, cytotoxic T lymphocytes; DC, dendritic cell; EpCAM, epithelial cell adhesion molecule; IgG, immunoglobulin G.

Techniques Used: Expressing, Incubation, Binding Assay, Staining, Negative Control, Isolation, Mouse Assay, CTL Assay, Concentration Assay

4) Product Images from "A Group A Streptococcus ADP-Ribosyltransferase Toxin Stimulates a Protective Interleukin 1β-Dependent Macrophage Immune Response"

Article Title: A Group A Streptococcus ADP-Ribosyltransferase Toxin Stimulates a Protective Interleukin 1β-Dependent Macrophage Immune Response

Journal: mBio

doi: 10.1128/mBio.00133-15

SpyA-deficient GAS bacteria evade macrophage killing. To measure total and intracellular killing, BMDMs isolated from C57BL/6 mice were activated through overnight incubation in DMEM supplemented with 2% FBS. (A) Total killing was measured by recovering total CFU of cells infected with GAS (AP) at an MOI of ~10 after 2 and 4 h. (B) Intracellular killing was assessed by recovering CFU from cells infected with GAS for 30 min followed by 100 µg/ml of gentamicin treatment (1 h); levels of internalized bacteria were monitored over 2 and 4 h in serum-free media. (C) BMDMs were infected with S. aureus RN4220 at an MOI of ~5, and intracellular killing was monitored at 4 and 6 h. (D) Transfected J774 cells were infected with Δ spyA GAS for 2 h in 2% FBS media for a total killing assay. Data shown are representative of the results of multiple repeats. Error bar; SEM. *, P
Figure Legend Snippet: SpyA-deficient GAS bacteria evade macrophage killing. To measure total and intracellular killing, BMDMs isolated from C57BL/6 mice were activated through overnight incubation in DMEM supplemented with 2% FBS. (A) Total killing was measured by recovering total CFU of cells infected with GAS (AP) at an MOI of ~10 after 2 and 4 h. (B) Intracellular killing was assessed by recovering CFU from cells infected with GAS for 30 min followed by 100 µg/ml of gentamicin treatment (1 h); levels of internalized bacteria were monitored over 2 and 4 h in serum-free media. (C) BMDMs were infected with S. aureus RN4220 at an MOI of ~5, and intracellular killing was monitored at 4 and 6 h. (D) Transfected J774 cells were infected with Δ spyA GAS for 2 h in 2% FBS media for a total killing assay. Data shown are representative of the results of multiple repeats. Error bar; SEM. *, P

Techniques Used: Isolation, Mouse Assay, Incubation, Infection, Transfection

5) Product Images from "Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct"

Article Title: Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00512.2016

Defb1 RNA in situ hybridization on wild-type C57BL/6 kidney section showing expression of Defb1 (purple, arrows; A ) and VATPase-E1 antibody (green, arrowhead; B ). C : overlay. *Cells positive for both VATPase (green) and Defb1 (purple). Magnification: ×60.
Figure Legend Snippet: Defb1 RNA in situ hybridization on wild-type C57BL/6 kidney section showing expression of Defb1 (purple, arrows; A ) and VATPase-E1 antibody (green, arrowhead; B ). C : overlay. *Cells positive for both VATPase (green) and Defb1 (purple). Magnification: ×60.

Techniques Used: RNA In Situ Hybridization, Expressing

6) Product Images from "Zika virus infection damages the testes in mice"

Article Title: Zika virus infection damages the testes in mice

Journal: Nature

doi: 10.1038/nature20556

Temporal loss of cellularity in the testis after ZIKV infection Seven week-old WT C57BL/6 mice were treated with 0.5 mg of anti-Ifnar1 at day -1 prior to subcutaneous inoculation of mouse-adapted ZIKV Dakar. Immunohistochemical analysis was performed on testis tissues collected from uninfected ( top panels ) or ZIKV-infected animals (days 7, 14 or 21 after infection; bottom panels ) at 20× ( left two images ) and 40× ( right image ) magnification. Staining was performed with antibodies against 3β-HSD (Leydig cells, left panels ), TRA98 (germ cells, middle panels ), and Lin28a (type A undifferentiated and type B spermatogonia, right panels ). Blue arrows indicate staining of Leydig cells ( left panels ), germ cells ( middle panels ), and spermatogonial stem cells ( right panels ). Red arrows indicate areas of virus-induced damage and loss of tissue integrity and specific cellularity. Scale bars = 200, 200, and 50 μm for the grouping of the three sets of images.
Figure Legend Snippet: Temporal loss of cellularity in the testis after ZIKV infection Seven week-old WT C57BL/6 mice were treated with 0.5 mg of anti-Ifnar1 at day -1 prior to subcutaneous inoculation of mouse-adapted ZIKV Dakar. Immunohistochemical analysis was performed on testis tissues collected from uninfected ( top panels ) or ZIKV-infected animals (days 7, 14 or 21 after infection; bottom panels ) at 20× ( left two images ) and 40× ( right image ) magnification. Staining was performed with antibodies against 3β-HSD (Leydig cells, left panels ), TRA98 (germ cells, middle panels ), and Lin28a (type A undifferentiated and type B spermatogonia, right panels ). Blue arrows indicate staining of Leydig cells ( left panels ), germ cells ( middle panels ), and spermatogonial stem cells ( right panels ). Red arrows indicate areas of virus-induced damage and loss of tissue integrity and specific cellularity. Scale bars = 200, 200, and 50 μm for the grouping of the three sets of images.

Techniques Used: Infection, Mouse Assay, Immunohistochemistry, Staining

Histology of the testes at day 28 after infection with ZIKV H/PF/2013 Seven week-old WT C57BL/6 mice were treated with PBS or anti-Ifnar1 at day -1 prior to subcutaneous inoculation in the footpad with 10 3 FFU of ZIKV H/PF/2013 or 10 6 FFU of DENV-2. Testes were collected at day 14 ( a ) or 28 ( b ) after infection and analyzed for viral RNA by qRT-PCR. Results are pooled from two independent biological experiments and each symbol represents data from an individual mouse. Bars indicate mean values. c . Histological analysis of PFA-fixed testis ( left panels ) and epididymis ( right panels ) tissues collected from uninfected or ZIKV-infected animals at day 28 at 20× ( left ) and 40× ( right ) magnification. Arrows indicate loss of germ cells and vacuoles in the testis (red), involution of epididymal lumens (yellow) with a mass of residual sperm (blue) and thickened epithelium (green). The images are representative of several independent experiments. Scale bars are indicated in the bottom right corner of the panels. Scale bars = 200 μm.
Figure Legend Snippet: Histology of the testes at day 28 after infection with ZIKV H/PF/2013 Seven week-old WT C57BL/6 mice were treated with PBS or anti-Ifnar1 at day -1 prior to subcutaneous inoculation in the footpad with 10 3 FFU of ZIKV H/PF/2013 or 10 6 FFU of DENV-2. Testes were collected at day 14 ( a ) or 28 ( b ) after infection and analyzed for viral RNA by qRT-PCR. Results are pooled from two independent biological experiments and each symbol represents data from an individual mouse. Bars indicate mean values. c . Histological analysis of PFA-fixed testis ( left panels ) and epididymis ( right panels ) tissues collected from uninfected or ZIKV-infected animals at day 28 at 20× ( left ) and 40× ( right ) magnification. Arrows indicate loss of germ cells and vacuoles in the testis (red), involution of epididymal lumens (yellow) with a mass of residual sperm (blue) and thickened epithelium (green). The images are representative of several independent experiments. Scale bars are indicated in the bottom right corner of the panels. Scale bars = 200 μm.

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR

ZIKV infection of the testis and epididymis at day 7 in Axl -/- and Rag1 -/- mice Seven week-old WT, Axl -/- , or Rag1 -/- C57BL/6 mice were treated with 0.5 mg of anti-Ifnar1 at day -1 prior to subcutaneous inoculation in the footpad with 10 6 FFU of mouse-adapted ZIKV Dakar. a . The indicated tissues were collected at day 7 after infection and analyzed for viral RNA by qRT-PCR. Dashed lines indicate limit of detection of the assays. b . ISH of testis from uninfected or ZIKV-infected WT and Axl -/- mice at day 7 with a ZIKV-specific probe. Dark blue arrows indicate Sertoli cells. ( Inset ) In sections from infected WT and Axl -/- mice, the cytoplasm of Sertoli cells is positive for ZIKV RNA (dark brown) with signal absent from prominent nuclei and nucleoli. Scale bar = 50 μm. c . Histology (H E, left two panels ) and ISH ( right two panels ) of testis from age-matched uninfected or ZIKV-infected (day 13) Rag1 -/- mice at 20× ( left ) and 40× ( right ) magnification for each pair. Scale bars = 200 and 50 μm. In H E stained testis sections arrows indicate loss of germ cells, and presence of multi-nucleated giant and necrotic cells from ZIKV-infected Rag1 -/- mice. In ISH, red and blue arrows indicate distribution of ZIKV RNA and Sertoli cells, respectively. d . IF ( three left and two right panels ) and IHC ( three middle ) staining of uninfected or ZIKV-infected (day 13) testis and epididymis from Rag1 -/- mice with antibodies to CD45, TRA98, ETV5, GATA4, Lin28a, 3b-HSD, F4/80 as described in Figure 1 and Extended Data Figure 2 . Colored arrows indicate staining for leukocytes (CD45, white), germ cells (TRA98, orange), Sertoli cells (GATA4, magenta), BTB (ETV5, green), type A undifferentiated and type B spermatogonia (Lin28a, black), and Leydig cells (3β-HSD, black) in respective panels. In the IHC staining panels with TRA98, red arrows indicate dying or dead germ cells and tubules without germ cells. White lines demarcate tubules in the seminiferous epithelium. Scale bars = 200 μm for IHC and 50 μm for IF. The images are representative from several different animals.
Figure Legend Snippet: ZIKV infection of the testis and epididymis at day 7 in Axl -/- and Rag1 -/- mice Seven week-old WT, Axl -/- , or Rag1 -/- C57BL/6 mice were treated with 0.5 mg of anti-Ifnar1 at day -1 prior to subcutaneous inoculation in the footpad with 10 6 FFU of mouse-adapted ZIKV Dakar. a . The indicated tissues were collected at day 7 after infection and analyzed for viral RNA by qRT-PCR. Dashed lines indicate limit of detection of the assays. b . ISH of testis from uninfected or ZIKV-infected WT and Axl -/- mice at day 7 with a ZIKV-specific probe. Dark blue arrows indicate Sertoli cells. ( Inset ) In sections from infected WT and Axl -/- mice, the cytoplasm of Sertoli cells is positive for ZIKV RNA (dark brown) with signal absent from prominent nuclei and nucleoli. Scale bar = 50 μm. c . Histology (H E, left two panels ) and ISH ( right two panels ) of testis from age-matched uninfected or ZIKV-infected (day 13) Rag1 -/- mice at 20× ( left ) and 40× ( right ) magnification for each pair. Scale bars = 200 and 50 μm. In H E stained testis sections arrows indicate loss of germ cells, and presence of multi-nucleated giant and necrotic cells from ZIKV-infected Rag1 -/- mice. In ISH, red and blue arrows indicate distribution of ZIKV RNA and Sertoli cells, respectively. d . IF ( three left and two right panels ) and IHC ( three middle ) staining of uninfected or ZIKV-infected (day 13) testis and epididymis from Rag1 -/- mice with antibodies to CD45, TRA98, ETV5, GATA4, Lin28a, 3b-HSD, F4/80 as described in Figure 1 and Extended Data Figure 2 . Colored arrows indicate staining for leukocytes (CD45, white), germ cells (TRA98, orange), Sertoli cells (GATA4, magenta), BTB (ETV5, green), type A undifferentiated and type B spermatogonia (Lin28a, black), and Leydig cells (3β-HSD, black) in respective panels. In the IHC staining panels with TRA98, red arrows indicate dying or dead germ cells and tubules without germ cells. White lines demarcate tubules in the seminiferous epithelium. Scale bars = 200 μm for IHC and 50 μm for IF. The images are representative from several different animals.

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, In Situ Hybridization, Staining, Immunohistochemistry

ZIKV infection of the testis and epididymis at ∼day 42. a–b Seven week-old WT C57BL/6 mice were treated with anti-Ifnar1 at day -1 prior to subcutaneous inoculation in the footpad with 10 5 FFU of mouse-adapted ZIKV Dakar. a . Testis ( left panels ) and epididymis ( right panels ) were collected at day 41 after infection or from age-matched uninfected mice, fixed with PFA, sectioned, stained with H E, and imaged at 20× ( left ) and 40× ( right ) magnification. Arrows show epididymal lumen void of sperm. The images are representative of sections from several independent animals. Scale bars are indicated in the bottom right corner of the panels. Scale bars = 200 and 50 μm. b . The indicated tissues and cells were collected at ∼day 42 after infection (days 41 ( n = 3), 42 ( n = 4), 43 ( n = 3), and 48 ( n =1)) and analyzed for viral RNA by qRT-PCR. Dashed line indicates the limit of detection of the assay. Results are pooled from two to three independent biological experiments and each symbol represents data from an individual mouse. Bars indicate median values.
Figure Legend Snippet: ZIKV infection of the testis and epididymis at ∼day 42. a–b Seven week-old WT C57BL/6 mice were treated with anti-Ifnar1 at day -1 prior to subcutaneous inoculation in the footpad with 10 5 FFU of mouse-adapted ZIKV Dakar. a . Testis ( left panels ) and epididymis ( right panels ) were collected at day 41 after infection or from age-matched uninfected mice, fixed with PFA, sectioned, stained with H E, and imaged at 20× ( left ) and 40× ( right ) magnification. Arrows show epididymal lumen void of sperm. The images are representative of sections from several independent animals. Scale bars are indicated in the bottom right corner of the panels. Scale bars = 200 and 50 μm. b . The indicated tissues and cells were collected at ∼day 42 after infection (days 41 ( n = 3), 42 ( n = 4), 43 ( n = 3), and 48 ( n =1)) and analyzed for viral RNA by qRT-PCR. Dashed line indicates the limit of detection of the assay. Results are pooled from two to three independent biological experiments and each symbol represents data from an individual mouse. Bars indicate median values.

Techniques Used: Infection, Mouse Assay, Staining, Quantitative RT-PCR

7) Product Images from "Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice"

Article Title: Viral FGARAT ORF75A promotes early events in lytic infection and gammaherpesvirus pathogenesis in mice

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006843

Intraperitoneal administration of MHV68 reveals a reactivation defect in the absence of ORF75A. C57BL/6 mice were infected at 1000 PFU by the intraperitoneal route with the indicated viruses. (A) Splenomegaly at 18 dpi with indicated viruses. Each symbol represents an individual mouse. Line indicates geometric mean titer. (B) Frequency of splenocytes harboring genomes 18 dpi. (C) Frequency of splenocytes spontaneously reactivating from latency 18 dpi. (D) Reactivation efficiency of splenocytes 18 dpi. (E) Frequency of PECs harboring genomes 18 dpi. (F) Frequency of PECs spontaneously reactivating from latency 18 dpi. For the limiting dilution analyses, curve fit lines were determined by nonlinear regression analysis. Using Poisson analysis, the intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were either positive for the viral genome or reactivating virus. Data is generated from 5 independent experiments with 4–5 mice per group for splenocyte data and 2 independent experiments with 4 mice per group for PEC data. ** p ≤ 0.005, *** p ≤ 0.0005, and **** p ≤ 0.00005; for A significance determined by two-way unpaired t-test; for B-F significance determined by two-way paired t-test.
Figure Legend Snippet: Intraperitoneal administration of MHV68 reveals a reactivation defect in the absence of ORF75A. C57BL/6 mice were infected at 1000 PFU by the intraperitoneal route with the indicated viruses. (A) Splenomegaly at 18 dpi with indicated viruses. Each symbol represents an individual mouse. Line indicates geometric mean titer. (B) Frequency of splenocytes harboring genomes 18 dpi. (C) Frequency of splenocytes spontaneously reactivating from latency 18 dpi. (D) Reactivation efficiency of splenocytes 18 dpi. (E) Frequency of PECs harboring genomes 18 dpi. (F) Frequency of PECs spontaneously reactivating from latency 18 dpi. For the limiting dilution analyses, curve fit lines were determined by nonlinear regression analysis. Using Poisson analysis, the intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were either positive for the viral genome or reactivating virus. Data is generated from 5 independent experiments with 4–5 mice per group for splenocyte data and 2 independent experiments with 4 mice per group for PEC data. ** p ≤ 0.005, *** p ≤ 0.0005, and **** p ≤ 0.00005; for A significance determined by two-way unpaired t-test; for B-F significance determined by two-way paired t-test.

Techniques Used: Mouse Assay, Infection, Generated

8) Product Images from "TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner"

Article Title: TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner

Journal: bioRxiv

doi: 10.1101/2019.12.13.875781

TF is not essential for virulence in type 1 interferon receptor deficient mice. A-B) Male and female 6-8 week old C57BL/6 Ifnar -/- mice were infected with WT SINV (n=14), 6K only (n=16), 4C (n=13), or 9C (n=13) via footpad inoculation (1e3 PFU). Survival was monitored (A) and viral load in serum was determined by plaque assay at 36 hours of infection in a subset of mice (B). This experiment was performed 3 independent times. N.d. indicates samples in which no virus was detected. C-D) Male and female 6-8 week old C57BL/6 Ifnar -/- mice were infected with WT SINV (n=10), 6K only (n=10), 4C (n=10), or 9C (n=10) via intracranial inoculation (1e3 PFU). Survival was monitored (A) and viral load in brains was determined by qRT-PCR at 24 hours of infection in a subset of mice (B). Virus load was normalized to the housekeeping gene, HPRT. Means +/- S.E.M. are shown for all viral quantitation along with data values for each mouse. The experiment was performed 2 times. Viral quantification data are expressed as mean +/- S.E.M. Statistics for survival curves were performed using Log-rank (Mantel-Cox) test. Statistics for viral quantification were performed using Student’s t-test comparing mutant virus to WT. ns = not significant.
Figure Legend Snippet: TF is not essential for virulence in type 1 interferon receptor deficient mice. A-B) Male and female 6-8 week old C57BL/6 Ifnar -/- mice were infected with WT SINV (n=14), 6K only (n=16), 4C (n=13), or 9C (n=13) via footpad inoculation (1e3 PFU). Survival was monitored (A) and viral load in serum was determined by plaque assay at 36 hours of infection in a subset of mice (B). This experiment was performed 3 independent times. N.d. indicates samples in which no virus was detected. C-D) Male and female 6-8 week old C57BL/6 Ifnar -/- mice were infected with WT SINV (n=10), 6K only (n=10), 4C (n=10), or 9C (n=10) via intracranial inoculation (1e3 PFU). Survival was monitored (A) and viral load in brains was determined by qRT-PCR at 24 hours of infection in a subset of mice (B). Virus load was normalized to the housekeeping gene, HPRT. Means +/- S.E.M. are shown for all viral quantitation along with data values for each mouse. The experiment was performed 2 times. Viral quantification data are expressed as mean +/- S.E.M. Statistics for survival curves were performed using Log-rank (Mantel-Cox) test. Statistics for viral quantification were performed using Student’s t-test comparing mutant virus to WT. ns = not significant.

Techniques Used: Mouse Assay, Infection, Plaque Assay, Quantitative RT-PCR, Quantitation Assay, Mutagenesis

TF antagonizes type 1 interferon responses in vitro and in vivo . A-B) Peritoneal macrophages were harvested from female C57BL/6 mice and plated in tissue culture for 48 hours to allow adherence. Cultures were washed to remove non-adherent cells and subsequently infected with WT Sindbis or 6K only (MOI =1). RNA was harvested at the indicated timepoints and virus (A) and interferon beta (B) were quantified by qRT-PCR. The housekeeping gene HPRT was used to normalize expression. Data are expressed as mean +/- S.E.M. of fold changes relative to the 2hpi timepoint of WT infected cells to allow pooling of three independent experiments. C) Male and female 3-4 week old C57BL/6 mice were infected with WT Sindbis (n=10) or 6K only (n=10) via intracranial inoculation (1e3 PFU). 24 hours post infection mice were euthanized, brains were harvested, and RNA was isolated. Levels of Sindbis virus and interferon beta RNA were quantified by qRT-PCR. Data are expressed as mean +/- S.E.M. of fold changes relative to the WT infected mice to allow pooling of two independent experiments. D) Peritoneal macrophages were harvested from female C57BL/6 mice and plated in tissue culture for 48 hours to allow adherence. Cultures were washed to remove non-adherent cells and subsequently infected with WT or 6K only SINV (MOI =1). At 24hpi, RNA was isolated and samples were assessed for two host transcripts with high turnover rates. Data for Csf-2 and Transferrin receptor p90 are expressed relative to HPRT values on the left y-axis, and HPRT Ct values are shown on the right y-axis. Statistics were performed using Student’s t-test in all cases.
Figure Legend Snippet: TF antagonizes type 1 interferon responses in vitro and in vivo . A-B) Peritoneal macrophages were harvested from female C57BL/6 mice and plated in tissue culture for 48 hours to allow adherence. Cultures were washed to remove non-adherent cells and subsequently infected with WT Sindbis or 6K only (MOI =1). RNA was harvested at the indicated timepoints and virus (A) and interferon beta (B) were quantified by qRT-PCR. The housekeeping gene HPRT was used to normalize expression. Data are expressed as mean +/- S.E.M. of fold changes relative to the 2hpi timepoint of WT infected cells to allow pooling of three independent experiments. C) Male and female 3-4 week old C57BL/6 mice were infected with WT Sindbis (n=10) or 6K only (n=10) via intracranial inoculation (1e3 PFU). 24 hours post infection mice were euthanized, brains were harvested, and RNA was isolated. Levels of Sindbis virus and interferon beta RNA were quantified by qRT-PCR. Data are expressed as mean +/- S.E.M. of fold changes relative to the WT infected mice to allow pooling of two independent experiments. D) Peritoneal macrophages were harvested from female C57BL/6 mice and plated in tissue culture for 48 hours to allow adherence. Cultures were washed to remove non-adherent cells and subsequently infected with WT or 6K only SINV (MOI =1). At 24hpi, RNA was isolated and samples were assessed for two host transcripts with high turnover rates. Data for Csf-2 and Transferrin receptor p90 are expressed relative to HPRT values on the left y-axis, and HPRT Ct values are shown on the right y-axis. Statistics were performed using Student’s t-test in all cases.

Techniques Used: In Vitro, In Vivo, Mouse Assay, Infection, Quantitative RT-PCR, Expressing, Isolation

Virulence of SINV is dependent on expression and palmitoylation of TF. A-B) Male and female 3-4 week old C57BL/6 mice were infected with WT SINV (n=19), 6K only (n=17), 4C (n=15), or 9C (n=10) via footpad inoculation (1e3 PFU). Survival was monitored (A) and infectious virus in serum was determined by plaque assay at 36 hours post infection in a subset of mice (B). Experiment was performed 3 times. A value followed by n.d. indicates the number of samples in which no virus was detected. C-D) Male and female 3-4 week old C57BL/6 mice were infected with WT SINV (n=17), 6K only (n=16), 4C (n=19), or 9C (n=14) via intracranial inoculation (1e3 PFU). Survival was monitored (C) and viral load in brains was determined by qRT-PCR at 24 hours post infection in a subset of mice (D). This experiment was performed 3 times. Virus load determinations were normalized to the housekeeping gene, HPRT. Means +/- S.E.M. are shown for all viral quantitation along with data values for each mouse. Statistics for survival curves were performed using Log-rank (Mantel-Cox) test. Statistics for viral quantification were performed using Student’s t-test comparing mutant virus to WT.
Figure Legend Snippet: Virulence of SINV is dependent on expression and palmitoylation of TF. A-B) Male and female 3-4 week old C57BL/6 mice were infected with WT SINV (n=19), 6K only (n=17), 4C (n=15), or 9C (n=10) via footpad inoculation (1e3 PFU). Survival was monitored (A) and infectious virus in serum was determined by plaque assay at 36 hours post infection in a subset of mice (B). Experiment was performed 3 times. A value followed by n.d. indicates the number of samples in which no virus was detected. C-D) Male and female 3-4 week old C57BL/6 mice were infected with WT SINV (n=17), 6K only (n=16), 4C (n=19), or 9C (n=14) via intracranial inoculation (1e3 PFU). Survival was monitored (C) and viral load in brains was determined by qRT-PCR at 24 hours post infection in a subset of mice (D). This experiment was performed 3 times. Virus load determinations were normalized to the housekeeping gene, HPRT. Means +/- S.E.M. are shown for all viral quantitation along with data values for each mouse. Statistics for survival curves were performed using Log-rank (Mantel-Cox) test. Statistics for viral quantification were performed using Student’s t-test comparing mutant virus to WT.

Techniques Used: Expressing, Mouse Assay, Infection, Plaque Assay, Quantitative RT-PCR, Quantitation Assay, Mutagenesis

9) Product Images from "Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer"

Article Title: Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1017668108

Detection of Wnt signaling by assay of β-galactosidase activity (blue) in frozen sections of the mucosa of C57BL/6 mice that harbor a lacZ transgene driven by a minimal fos promoter under regulation of three tandem Tcf4 binding sites. In this case, mice were fed the three diets from weaning for 3 mo.
Figure Legend Snippet: Detection of Wnt signaling by assay of β-galactosidase activity (blue) in frozen sections of the mucosa of C57BL/6 mice that harbor a lacZ transgene driven by a minimal fos promoter under regulation of three tandem Tcf4 binding sites. In this case, mice were fed the three diets from weaning for 3 mo.

Techniques Used: Activity Assay, Mouse Assay, Binding Assay

10) Product Images from "Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL"

Article Title: Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160049

Loss of Ikaros tumor suppression in Ikzf1 ΔF4/ΔF4 mutant mice results in an increase in active cell cycle and aggressive leukemia in a nonirradiated in vivo model . (A–C) BM from WT, Ikzf1 ΔF1/ΔF1 , and Ikzf1 ΔF4/ΔF4 mutant mice were transduced with BCR-ABL1-p185-IRES-YFP and grown in vitro on BM stroma–derived feeder layers. (A) Cell cycle flow cytometry analysis was performed by Hoechst incorporation, and one representative experiment (at day 14 of cell culture) is shown. (B) Cells were harvested on different days of in vitro cell culture and protein was extracted for Western blot analysis of Cyclin D1, Cdk6, p16, and p21, with ERK as loading control. Space indicates that intervening lanes have been spliced out. (C) RNA was isolated from the in vitro cultures, cDNA was prepared, and mRNA for Ccnd1 , Cdk6 , p16 , and p21 was analyzed by quantitative real-time PCR. Ubc was used as a reference gene. (D) Schematic of the experimental set up for the in vivo transplantation assay. BM cells from WT and Ikzf1 ΔF4/ΔF4 mutant mice were transduced and cultured in vitro as in A–C for 7 d before i.v. injection into nonirradiated immunocompetent WT c57BL/6 recipient mice. Animals were monitored and euthanized upon signs of leukemia development. (E) Kaplan-Meyer curve of cohorts receiving 10 5 (left) or 10 4 (right) BCR-ABL1–transformed cells. (A–C) are representative of three independent experiments and (E) represents one experiment with five recipient mice for each of the four cohorts. The result in right panel was repeated in a separate independent experiment displayed in Fig. 6 F .
Figure Legend Snippet: Loss of Ikaros tumor suppression in Ikzf1 ΔF4/ΔF4 mutant mice results in an increase in active cell cycle and aggressive leukemia in a nonirradiated in vivo model . (A–C) BM from WT, Ikzf1 ΔF1/ΔF1 , and Ikzf1 ΔF4/ΔF4 mutant mice were transduced with BCR-ABL1-p185-IRES-YFP and grown in vitro on BM stroma–derived feeder layers. (A) Cell cycle flow cytometry analysis was performed by Hoechst incorporation, and one representative experiment (at day 14 of cell culture) is shown. (B) Cells were harvested on different days of in vitro cell culture and protein was extracted for Western blot analysis of Cyclin D1, Cdk6, p16, and p21, with ERK as loading control. Space indicates that intervening lanes have been spliced out. (C) RNA was isolated from the in vitro cultures, cDNA was prepared, and mRNA for Ccnd1 , Cdk6 , p16 , and p21 was analyzed by quantitative real-time PCR. Ubc was used as a reference gene. (D) Schematic of the experimental set up for the in vivo transplantation assay. BM cells from WT and Ikzf1 ΔF4/ΔF4 mutant mice were transduced and cultured in vitro as in A–C for 7 d before i.v. injection into nonirradiated immunocompetent WT c57BL/6 recipient mice. Animals were monitored and euthanized upon signs of leukemia development. (E) Kaplan-Meyer curve of cohorts receiving 10 5 (left) or 10 4 (right) BCR-ABL1–transformed cells. (A–C) are representative of three independent experiments and (E) represents one experiment with five recipient mice for each of the four cohorts. The result in right panel was repeated in a separate independent experiment displayed in Fig. 6 F .

Techniques Used: Mutagenesis, Mouse Assay, In Vivo, Transduction, In Vitro, Derivative Assay, Flow Cytometry, Cytometry, Cell Culture, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Transplantation Assay, Injection, Transformation Assay

Kit is a ZnF4-dependent Ikaros target gene that confers growth advantage and correlates with aggressive leukemia. (A) Schematic diagram of relevant cell surface markers expressed on developing B cells. (B) ChIP-seq tracks of Ikaros and input from mouse pro-B cells ( Bossen et al., 2015 ) demonstrates binding of Ikaros at the Kit promoter. (C) Retroviral overexpression of Ikaros isoforms containing different subsets of the DNA-binding zinc fingers (IRES-GFP) in Ikzf1 null , EBF1-rescued noncommitted pro–B cells ( Reynaud et al., 2008 ). GFP and c-Kit expression was analyzed by flow cytometry. (D) Mouse pre–B ALL cells from WT and Ikzf1 ΔF4/ΔF4 mutant mice were created and cultured as described in Fig. 1 A . Cell growth with and without c-Kit blocking Ab (ACK45; 10 µg/ml) was measured by daily cell count. (C and D) One representative of two independent experiments. (E) Day 7 cultures of mouse pre–B ALL cells from WT and Ikzf1 ΔF4/ΔF4 mutant mice were sorted as indicated based on c-Kit expression. (F) Sorted cells from E or bulk unsorted day 7 cultures were i.v. injected into nonirradiated, immunocompetent WT C57BL/6 mice. Mice were monitored and euthanized upon development of leukemia. Leukemia was confirmed by necropsy and flow cytometry analysis, and survival of indicated cohorts is represented by Kaplan-Meyer curve.
Figure Legend Snippet: Kit is a ZnF4-dependent Ikaros target gene that confers growth advantage and correlates with aggressive leukemia. (A) Schematic diagram of relevant cell surface markers expressed on developing B cells. (B) ChIP-seq tracks of Ikaros and input from mouse pro-B cells ( Bossen et al., 2015 ) demonstrates binding of Ikaros at the Kit promoter. (C) Retroviral overexpression of Ikaros isoforms containing different subsets of the DNA-binding zinc fingers (IRES-GFP) in Ikzf1 null , EBF1-rescued noncommitted pro–B cells ( Reynaud et al., 2008 ). GFP and c-Kit expression was analyzed by flow cytometry. (D) Mouse pre–B ALL cells from WT and Ikzf1 ΔF4/ΔF4 mutant mice were created and cultured as described in Fig. 1 A . Cell growth with and without c-Kit blocking Ab (ACK45; 10 µg/ml) was measured by daily cell count. (C and D) One representative of two independent experiments. (E) Day 7 cultures of mouse pre–B ALL cells from WT and Ikzf1 ΔF4/ΔF4 mutant mice were sorted as indicated based on c-Kit expression. (F) Sorted cells from E or bulk unsorted day 7 cultures were i.v. injected into nonirradiated, immunocompetent WT C57BL/6 mice. Mice were monitored and euthanized upon development of leukemia. Leukemia was confirmed by necropsy and flow cytometry analysis, and survival of indicated cohorts is represented by Kaplan-Meyer curve.

Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Over Expression, Zinc-Fingers, Expressing, Flow Cytometry, Cytometry, Mutagenesis, Mouse Assay, Cell Culture, Blocking Assay, Cell Counting, Injection

11) Product Images from "CFTR-PTEN–dependent mitochondrial metabolic dysfunction promotes Pseudomonas aeruginosa airway infection"

Article Title: CFTR-PTEN–dependent mitochondrial metabolic dysfunction promotes Pseudomonas aeruginosa airway infection

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aav4634

CF P. aeruginosa isolates persist in the airway. C57Bl/6 WT mice were intranasally infected with 10 7 CFUs of either PAO1, single CF isolates, a mixture of two, or all 17 strains together. ( A and B ) Mouse survival (five mice per group pooled from n = 2). ( C ) P. aeruginosa burden in BAL (four to seven mice per group pooled from n = 3). ( D to F ) Monocytes, neutrophils, and alveolar macrophages found in BAL 24 hours after infection (four to six mice per group pooled from n = 3). Cftr ΔF508/ΔF508 (gut-corrected) mice were intranasally infected with a mix of 10 7 CFUs of all 17 CF P. aeruginosa isolates. ( G ) Succinate in BAL (three to eight mice per group pooled from n = 3). ( H to J ) Neutrophils, monocytes, and alveolar macrophages found in BAL 48 hours after infection (eight mice per group pooled from n = 3). ( K and L ) P. aeruginosa burden in BAL and lungs (eight mice per group pooled from n = 3). Ptenl −/− mice were intranasally infected with a mix of 10 7 CFUs of all 17 CF P. aeruginosa isolates (three to six mice per group pooled from n = 3). The following were analyzed: ( M ) succinate in BAL; ( N ) IL-1β in BAL; ( O to Q ) monocytes, neutrophils, and alveolar macrophages found in BAL 24 hours after infection, ( R and S ) P. aeruginosa burden in BAL and lungs 24 hours after infection. Data are shown as means ± SEM. (A and B) Kaplan-Meier; (C) two-way ANOVA; (D to G and M to S) one-way ANOVA; (H to L) Student’s t test. **** P
Figure Legend Snippet: CF P. aeruginosa isolates persist in the airway. C57Bl/6 WT mice were intranasally infected with 10 7 CFUs of either PAO1, single CF isolates, a mixture of two, or all 17 strains together. ( A and B ) Mouse survival (five mice per group pooled from n = 2). ( C ) P. aeruginosa burden in BAL (four to seven mice per group pooled from n = 3). ( D to F ) Monocytes, neutrophils, and alveolar macrophages found in BAL 24 hours after infection (four to six mice per group pooled from n = 3). Cftr ΔF508/ΔF508 (gut-corrected) mice were intranasally infected with a mix of 10 7 CFUs of all 17 CF P. aeruginosa isolates. ( G ) Succinate in BAL (three to eight mice per group pooled from n = 3). ( H to J ) Neutrophils, monocytes, and alveolar macrophages found in BAL 48 hours after infection (eight mice per group pooled from n = 3). ( K and L ) P. aeruginosa burden in BAL and lungs (eight mice per group pooled from n = 3). Ptenl −/− mice were intranasally infected with a mix of 10 7 CFUs of all 17 CF P. aeruginosa isolates (three to six mice per group pooled from n = 3). The following were analyzed: ( M ) succinate in BAL; ( N ) IL-1β in BAL; ( O to Q ) monocytes, neutrophils, and alveolar macrophages found in BAL 24 hours after infection, ( R and S ) P. aeruginosa burden in BAL and lungs 24 hours after infection. Data are shown as means ± SEM. (A and B) Kaplan-Meier; (C) two-way ANOVA; (D to G and M to S) one-way ANOVA; (H to L) Student’s t test. **** P

Techniques Used: Mouse Assay, Infection

12) Product Images from "Local Inhibition of Complement Improves Mesenchymal Stem Cell Viability and Function After Administration"

Article Title: Local Inhibition of Complement Improves Mesenchymal Stem Cell Viability and Function After Administration

Journal: Molecular Therapy

doi: 10.1038/mt.2016.142

Heparin-painted mesenchymal stem cells (MSCs) (Hep-MSCs) are more effective in inhibiting the proliferation of antigen-specific T cells in vivo than mock-painted MSCs (Ctrl-MSCs) . Naive C57BL/6 mice were infused with 1 × 10 6 purified
Figure Legend Snippet: Heparin-painted mesenchymal stem cells (MSCs) (Hep-MSCs) are more effective in inhibiting the proliferation of antigen-specific T cells in vivo than mock-painted MSCs (Ctrl-MSCs) . Naive C57BL/6 mice were infused with 1 × 10 6 purified

Techniques Used: In Vivo, Mouse Assay, Purification

13) Product Images from "T Lymphocyte–Specific Activation of Nrf2 Protects from AKI"

Article Title: T Lymphocyte–Specific Activation of Nrf2 Protects from AKI

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2014100978

Effect of adoptive transfer of CD4-Keap1-KO T cells into WT (C57BL/6) mice ( n =7–10). (A) The success of adoptive transfer is confirmed by establishing the presence of CFSE-labeled T cells in peripheral blood of WT recipients before inducing AKI.
Figure Legend Snippet: Effect of adoptive transfer of CD4-Keap1-KO T cells into WT (C57BL/6) mice ( n =7–10). (A) The success of adoptive transfer is confirmed by establishing the presence of CFSE-labeled T cells in peripheral blood of WT recipients before inducing AKI.

Techniques Used: Adoptive Transfer Assay, Mouse Assay, Labeling

14) Product Images from "A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses"

Article Title: A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006258

Stat2 -/- mice support ZIKV infection and recapitulate ZIKV pathogenesis and disease. Five to six week old female WT, Stat2 -/- and Ifnar1 -/- C57BL/6 mice (n = 5) were injected with 1,000 PFU of ZIKV strain MR766 by the subcutaneous route in the footpad. (A) Mice were weighed daily and weights are expressed as percentage of body weight prior to infection. Results shown are the mean ± standard error of the mean (SEM). Data are censored at 6 days after infection, as mice in the Stat2 -/- group succumbed to infection. (B) In a parallel set of mice (n = 5) lethality was monitored for 14 days. (C) Clinical signs of ZIKV infection were monitored every day in the group of animals used for survival analysis (n = 5). Clinical signs are abbreviated as HB (hunched back, reduced motility), HL1 (one hind limb paralysis), HL2 (both hind limbs paralyzed), FL (one or both front limbs paralyzed), Endpoint (loss of 25% of initial body weight or dead). The percentage of each group of mice displaying the indicated signs is shown. (D) From the group of mice used to monitor body weight loss (n = 5, day 6 post infection), indicated organs were harvested and ZIKV RNA levels were measured by qRT PCR as described in methods. Error bars represent mean ±standard deviation (SD). Y axis starts at the limit of detection of the assay.
Figure Legend Snippet: Stat2 -/- mice support ZIKV infection and recapitulate ZIKV pathogenesis and disease. Five to six week old female WT, Stat2 -/- and Ifnar1 -/- C57BL/6 mice (n = 5) were injected with 1,000 PFU of ZIKV strain MR766 by the subcutaneous route in the footpad. (A) Mice were weighed daily and weights are expressed as percentage of body weight prior to infection. Results shown are the mean ± standard error of the mean (SEM). Data are censored at 6 days after infection, as mice in the Stat2 -/- group succumbed to infection. (B) In a parallel set of mice (n = 5) lethality was monitored for 14 days. (C) Clinical signs of ZIKV infection were monitored every day in the group of animals used for survival analysis (n = 5). Clinical signs are abbreviated as HB (hunched back, reduced motility), HL1 (one hind limb paralysis), HL2 (both hind limbs paralyzed), FL (one or both front limbs paralyzed), Endpoint (loss of 25% of initial body weight or dead). The percentage of each group of mice displaying the indicated signs is shown. (D) From the group of mice used to monitor body weight loss (n = 5, day 6 post infection), indicated organs were harvested and ZIKV RNA levels were measured by qRT PCR as described in methods. Error bars represent mean ±standard deviation (SD). Y axis starts at the limit of detection of the assay.

Techniques Used: Mouse Assay, Infection, Injection, Quantitative RT-PCR, Standard Deviation

Comparison of weight loss and mortality induced by African and Asian ZIKV strains in mice. Five to six week old male WT, Stat2 -/- and Ifnar1 -/- C57BL/6 mice (n = 5) were injected with 1,000 PFU of the indicated ZIKV strain by the subcutaneous route in the footpad. (A, B) Mice were weighed daily and weights are expressed as percentage of body weight prior to infection. Results shown are the mean ± standard error of the mean (SEM). (A) Data are censored at 6 days after infection, as some mice died. (C, D) In a parallel group, male mice (n = 5) of the indicated genotype were infected with indicated ZIKV strains under similar conditions and data were combined with body weight loss group for survival analysis (total n = 10). ZIKV strains are abbreviated as UG (Uganda), SN (Senegal), ML (Malaysia), CB (Cambodia) and PR (Puerto Rico).
Figure Legend Snippet: Comparison of weight loss and mortality induced by African and Asian ZIKV strains in mice. Five to six week old male WT, Stat2 -/- and Ifnar1 -/- C57BL/6 mice (n = 5) were injected with 1,000 PFU of the indicated ZIKV strain by the subcutaneous route in the footpad. (A, B) Mice were weighed daily and weights are expressed as percentage of body weight prior to infection. Results shown are the mean ± standard error of the mean (SEM). (A) Data are censored at 6 days after infection, as some mice died. (C, D) In a parallel group, male mice (n = 5) of the indicated genotype were infected with indicated ZIKV strains under similar conditions and data were combined with body weight loss group for survival analysis (total n = 10). ZIKV strains are abbreviated as UG (Uganda), SN (Senegal), ML (Malaysia), CB (Cambodia) and PR (Puerto Rico).

Techniques Used: Mouse Assay, Injection, Infection

15) Product Images from "SOCS1 is a negative regulator of metabolic reprogramming during sepsis"

Article Title: SOCS1 is a negative regulator of metabolic reprogramming during sepsis

Journal: JCI Insight

doi: 10.1172/jci.insight.92530

Inhibition of SOCS1 increases bacterial burden and organ damage during sepsis. ( A ) SOCS1 mRNA expression levels in the blood of septic pediatric patients and normal controls, as determined by qPCR (septic shock, n = 180 and normal controls, n = 52); ANOVA, and corrections for multiple comparisons were performed using a Benjamini-Hochberg false discovery rate of 5%. ( B ) Socs1 mRNA expression levels in C57BL/6 mouse peritoneal cells 18 hours after cecal ligation and puncture–induced (CLP-induced) sepsis, as determined by qPCR ( n = 8 mice/group, t test, Mann-Whitney U test); * P
Figure Legend Snippet: Inhibition of SOCS1 increases bacterial burden and organ damage during sepsis. ( A ) SOCS1 mRNA expression levels in the blood of septic pediatric patients and normal controls, as determined by qPCR (septic shock, n = 180 and normal controls, n = 52); ANOVA, and corrections for multiple comparisons were performed using a Benjamini-Hochberg false discovery rate of 5%. ( B ) Socs1 mRNA expression levels in C57BL/6 mouse peritoneal cells 18 hours after cecal ligation and puncture–induced (CLP-induced) sepsis, as determined by qPCR ( n = 8 mice/group, t test, Mann-Whitney U test); * P

Techniques Used: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Ligation, Mouse Assay, MANN-WHITNEY

16) Product Images from "Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis"

Article Title: Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.40

Localization of HMGB1 in lysosome induces lysosomal destabilization and cathepsin B (CatB) activation. ( a ) WT AMs were treated with HMGB1-EGFP (green, 100 nmol/l) for 5 min, 15 min, 30 min, 1 h, 3 h, or 6 h, then immunofluorescence of early endosome antigen 1 (red) were detected. Original magnification × 600; results are representative of three independent experiments. ( b ) AMs were incubated with both HMGB1-EGFP (green, 100 nmol/l) and LysoTracker Red lysosome dye (red) for 5 min, 30 min, 1 h, 3 h, 6 h, or 12 h. Co-localization of HMGB1 and lysosome was detected using confocal microscopy. Original magnification × 600; results are representative of three independent experiments. ( c ) AMs were treated with HMGB1 (50 nmol/l) for 0–12 h followed by incubation with DQ ovalbumin (red) for 1 h to visualize lysosome integrity using confocal microscopy. Original magnification × 600; results are representative of three independent experiments. ( d ) AMs isolated from C57BL/6 (WT) or RAGE −/− mice were incubated with HMGB1 (50 nmol/l) for 30 min in the absence or presence of dynasore (30 μ g/ml) or DMSO (0.3%). The cells were then stained with Magic Red CatB detection reagent (red) to visualize activated CatB under confocal microscopy. Original magnification × 600; results are representative of three independent experiments. The insets show higher magnification views. ( e – g ) Macrophages were stained by Magic Red CatB detection reagent to detect the activation of CatB. The mean fluorescence intensity (MFI) of activated CatB was detected and analyzed using confocal microscopy and OLYMPUS Fluoview Ver.1.7c system. ( e ) WT AMs were stimulated with HMGB1 (50 nmol/l) for 0–12 h (mean and S.D., n =5. * P
Figure Legend Snippet: Localization of HMGB1 in lysosome induces lysosomal destabilization and cathepsin B (CatB) activation. ( a ) WT AMs were treated with HMGB1-EGFP (green, 100 nmol/l) for 5 min, 15 min, 30 min, 1 h, 3 h, or 6 h, then immunofluorescence of early endosome antigen 1 (red) were detected. Original magnification × 600; results are representative of three independent experiments. ( b ) AMs were incubated with both HMGB1-EGFP (green, 100 nmol/l) and LysoTracker Red lysosome dye (red) for 5 min, 30 min, 1 h, 3 h, 6 h, or 12 h. Co-localization of HMGB1 and lysosome was detected using confocal microscopy. Original magnification × 600; results are representative of three independent experiments. ( c ) AMs were treated with HMGB1 (50 nmol/l) for 0–12 h followed by incubation with DQ ovalbumin (red) for 1 h to visualize lysosome integrity using confocal microscopy. Original magnification × 600; results are representative of three independent experiments. ( d ) AMs isolated from C57BL/6 (WT) or RAGE −/− mice were incubated with HMGB1 (50 nmol/l) for 30 min in the absence or presence of dynasore (30 μ g/ml) or DMSO (0.3%). The cells were then stained with Magic Red CatB detection reagent (red) to visualize activated CatB under confocal microscopy. Original magnification × 600; results are representative of three independent experiments. The insets show higher magnification views. ( e – g ) Macrophages were stained by Magic Red CatB detection reagent to detect the activation of CatB. The mean fluorescence intensity (MFI) of activated CatB was detected and analyzed using confocal microscopy and OLYMPUS Fluoview Ver.1.7c system. ( e ) WT AMs were stimulated with HMGB1 (50 nmol/l) for 0–12 h (mean and S.D., n =5. * P

Techniques Used: Activation Assay, Affinity Magnetic Separation, Immunofluorescence, Incubation, Confocal Microscopy, Isolation, Mouse Assay, Staining, Fluorescence

Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and dynamin inhibitor dynasore (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P
Figure Legend Snippet: Macrophage endocytosis of HMGB1 is mediated by RAGE-dependent pathway. ( a ) Confocal microscopy of alveolar macrophages (AMs) that were isolated from C57BL/6 (wild type, WT) mice and incubated with recombinant protein HMGB1-EGFP (100 nmol/l) or EGFP for 0–30 min. Original magnification × 600; results are representative of three independent experiments. ( b ) Confocal microscopy of AMs that were isolated from WT mice and incubated with 0–100 nmol/l HMGB1-EGFP for 30 min. Original magnification × 600; results are representative of three independent experiments. ( c ) Confocal microscopy of AMs that were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. Original magnification × 600; results are representative of three independent experiments. ( d ) Changes in average number of intracellular EGFP-tagged protein particles in AMs, which were calculated using confocal microscopy program. The AMs were isolated from WT, TLR2 −/− , TLR4 −/− , or RAGE −/− mice and incubated with HMGB1-EGFP (100 nmol/l) for 30 min. In some experiments, WT AMs were incubated with HMGB1-EGFP and dynamin inhibitor dynasore (30 μ g/ml) for 30 min. The graph shows the mean and S.D., n =9. * P

Techniques Used: Confocal Microscopy, Affinity Magnetic Separation, Isolation, Mouse Assay, Incubation, Recombinant

HMGB1 induces macrophage pyroptosis in mouse endotoxemia model. ( a ) C57BL/6 mice were injected with LPS (20 mg/kg BW) or normal saline (NS) intraperitoneally (i.p.). AMs were isolated at 18 h after LPS injection and stained with FLICA caspase-1 fluorescent reagent (green), Cell Death Reagent (red), and Hoechst (blue). Images were obtained by confocal microscopy (original magnification × 600). ( b ) WT or RAGE −/− mice were injected with LPS or NS i.p. Neutralizing anti-HMGB1 IgY (SHINO-TEST Corporation) or control nonimmune IgY (Fitzgerald, North Acton, MA) in a dose of 2 mg/kg BW was administered to some animals 30 min before LPS i.p. injection. Blood monocytes were collected at 18 h after LPS and stained with Cell Death reagent (TMR red) and FLICA caspase-1 fluorescent reagent (AF488), and double-stained pyroptotic monocytes were detected by flow cytometry. Results are representative of three independent experiments. The graph shows the mean and S.D., n =3. * P
Figure Legend Snippet: HMGB1 induces macrophage pyroptosis in mouse endotoxemia model. ( a ) C57BL/6 mice were injected with LPS (20 mg/kg BW) or normal saline (NS) intraperitoneally (i.p.). AMs were isolated at 18 h after LPS injection and stained with FLICA caspase-1 fluorescent reagent (green), Cell Death Reagent (red), and Hoechst (blue). Images were obtained by confocal microscopy (original magnification × 600). ( b ) WT or RAGE −/− mice were injected with LPS or NS i.p. Neutralizing anti-HMGB1 IgY (SHINO-TEST Corporation) or control nonimmune IgY (Fitzgerald, North Acton, MA) in a dose of 2 mg/kg BW was administered to some animals 30 min before LPS i.p. injection. Blood monocytes were collected at 18 h after LPS and stained with Cell Death reagent (TMR red) and FLICA caspase-1 fluorescent reagent (AF488), and double-stained pyroptotic monocytes were detected by flow cytometry. Results are representative of three independent experiments. The graph shows the mean and S.D., n =3. * P

Techniques Used: Mouse Assay, Injection, Affinity Magnetic Separation, Isolation, Staining, Confocal Microscopy, Flow Cytometry, Cytometry

17) Product Images from "L-Myc expression by dendritic cells is required for optimal T-cell priming"

Article Title: L-Myc expression by dendritic cells is required for optimal T-cell priming

Journal: Nature

doi: 10.1038/nature12967

Mycl1 expression within the immune system is restricted to dendritic cells a , Shown is the percent of BrdU + cells within each indicated lineage from WT mice 4 hours after BrdU administration. Data are from 2 independent experiments (bars, SD; n = 9 mice per group, one-way analysis of variance (ANOVA) Tukey's post hoc test). b , Shown are single-color histograms of DAPI and Ki67 expression for indicated cell types from the spleen of WT mice. Dotted lines represent isotype control staining. Numbers represent percent of cells in the indicated gate. Data are representative of 3 independent experiments. c , Shown are two-color histograms of CD11c and SiglecH expression for all live BM cells from WT mice, or B220 and MHCII expression for CD11c + SiglecH + cells, which were evaluated for DAPI expression (bottom panels). d , Relative Mycl1 mRNA expression was determined by quantitative real-time PCR (qPCR) in pDCs isolated from BM and other indicated cell types isolated from the spleen of WT mice. Shown is a graph of Mycl1 values normalized to Hprt values (bar, SD; n=3 biological replicates per cell type). e , CMP and CDP populations described in Methods Summary were analyzed by gene expression microarray. Shown are transcription factor probe sets with normalized linear expression value in the CDP greater than 100. The horizontal axis indicates the ratio of gene expression in the CMP compared to the CDP, and the vertical axis ind icates the ratio of the mean expression in CD8α + cDCs, CD11b + cDCs and pDCs compared to a tissue panel excluding DCs (described in Methods). f, Relative Mycl1 mRNA expression was determined by qPCR for indicated mice. Shown is a graph of Mycl1 values normalized to Hprt values (bar, SD; n=3 biological replicates per cell type). g , Splenocytes from WT and Mycl1 gfp/gfp mice on the C57BL/6 genetic background were stained for analysis, and pDCs (CD11c int SiglecH + ), CD8α + DCs (CD11c + MHCII + CD8α + CD205 + SiglecH − ), and CD8α − DCs (CD11c + MHCII + CD8α − CD205-SiglecH − ) were identified. Shown are two-color histograms of CD11c and SiglecH expression, MHCII and CD11c expression, and CD8α and CD205 expression for pre-gated cells as indicated above the plots. Numbers represent percent of cells in the indicated gate. Data are representative of at least 5 independent experiments. h , Relative Myc mRNA expression was determined by qPCR for indicated mice. Shown is a graph of Myc values normalized to Hprt values (bar, SD; n=3 biological replicates per cell type, Student's t -test). *, p
Figure Legend Snippet: Mycl1 expression within the immune system is restricted to dendritic cells a , Shown is the percent of BrdU + cells within each indicated lineage from WT mice 4 hours after BrdU administration. Data are from 2 independent experiments (bars, SD; n = 9 mice per group, one-way analysis of variance (ANOVA) Tukey's post hoc test). b , Shown are single-color histograms of DAPI and Ki67 expression for indicated cell types from the spleen of WT mice. Dotted lines represent isotype control staining. Numbers represent percent of cells in the indicated gate. Data are representative of 3 independent experiments. c , Shown are two-color histograms of CD11c and SiglecH expression for all live BM cells from WT mice, or B220 and MHCII expression for CD11c + SiglecH + cells, which were evaluated for DAPI expression (bottom panels). d , Relative Mycl1 mRNA expression was determined by quantitative real-time PCR (qPCR) in pDCs isolated from BM and other indicated cell types isolated from the spleen of WT mice. Shown is a graph of Mycl1 values normalized to Hprt values (bar, SD; n=3 biological replicates per cell type). e , CMP and CDP populations described in Methods Summary were analyzed by gene expression microarray. Shown are transcription factor probe sets with normalized linear expression value in the CDP greater than 100. The horizontal axis indicates the ratio of gene expression in the CMP compared to the CDP, and the vertical axis ind icates the ratio of the mean expression in CD8α + cDCs, CD11b + cDCs and pDCs compared to a tissue panel excluding DCs (described in Methods). f, Relative Mycl1 mRNA expression was determined by qPCR for indicated mice. Shown is a graph of Mycl1 values normalized to Hprt values (bar, SD; n=3 biological replicates per cell type). g , Splenocytes from WT and Mycl1 gfp/gfp mice on the C57BL/6 genetic background were stained for analysis, and pDCs (CD11c int SiglecH + ), CD8α + DCs (CD11c + MHCII + CD8α + CD205 + SiglecH − ), and CD8α − DCs (CD11c + MHCII + CD8α − CD205-SiglecH − ) were identified. Shown are two-color histograms of CD11c and SiglecH expression, MHCII and CD11c expression, and CD8α and CD205 expression for pre-gated cells as indicated above the plots. Numbers represent percent of cells in the indicated gate. Data are representative of at least 5 independent experiments. h , Relative Myc mRNA expression was determined by qPCR for indicated mice. Shown is a graph of Myc values normalized to Hprt values (bar, SD; n=3 biological replicates per cell type, Student's t -test). *, p

Techniques Used: Expressing, Mouse Assay, Staining, Real-time Polymerase Chain Reaction, Isolation, Microarray

IRF8 regulates the expression of Mycl1 in multiple dendritic cell lineages a , (left panels) BM cells and splenocytes from Mycl1 +/+ Irf8 +/+ , Mycl1 +/gfp Irf8 +/+ , Mycl1 +/+ Irf8 R294C , and Mycl1 +/gfp Irf8 R294C mice were stained for analysis. Shown are two-color histograms of GFP and CD11c expression. Numbers represent the percent of cells in the indicate gates. Data are representative of 3 independent experiments. (right panels) Ly6C + CD11b + BM monocytes were isolated by cell sorting from Mycl1 +/+ Irf8 +/+ (black line), Mycl1 +/+ Irf8 R294C (grey line), Mycl1 +/gfp Irf8 R294C (light green line), Mycl1 +/gfp Irf8 +/+ mice (dark green line) and differentiated for 4 days either with GM-CSF (left panel) or with GM-CSF and IL-4 (right panel). Shown are single-color histograms of GFP expression for pre-gated live CD11c + cells from the indicated genotypes. b , Splenocytes from WT (top panels) and Irf8 R294C (bottom panels) mice on the C57BL/6 genetic background were stained for analysis. Shown are two-color histograms of CD11c and SiglecH expression (left panels), and CD11b and CD205 expression (right panels), for cells pre-gated as either B220 + CD11b − or CD11c + MHCII + B220 − . pDCs were gated as CD11c + SiglecH + B220 + CD11b − . Numbers represent the percent of cells in the indicated gates. Data are representative of 2 independent experiments. c , Ly6C + BM monocytes were isolated by cell sorting from Mycl1 +/+ Irf8 +/+ , Mycl1 +/+ Irf8 R294C , Mycl1 +/gfp Irf8 R294C , and Mycl1 +/gfp Irf8 +/+ mice. Monocytes were cultured either with GM-CSF or with GM-CSF and IL-4 for 4 days and then stained for analysis. Shown are two-color histograms of MHCII and CD11c expression for differentiated monocytes. Numbers represent the percent of cells in the quadrant gate. Data are representative of 2 independent experiments. d , Phoenix-E cells were transfected with murine stem cell virus (MSCV) retroviral plasmids expressing the mutant human estrogen receptor (ER T2 ), murine Myc (c-MYC), murine Mycl1 (L-MYC), and a fusion between Mycl1 and the mutant human estrogen receptor (L-MYC-ER T2 ). Whole-cell extracts were prepared 2 days after transfection and Western blot analysis was performed. Shown are blots probed for L-MYC (left, α-L-MYC) and c-MYC (right, α-c-MYC) for the indicated transfections. Blots were stripped and re-probed for GFP (bottom, α-GFP). e , IRF8 DNA binding was assayed by ChIP-Seq. Shown is a histogram of normalized reads per bp per peak within a 4-kb window centered on transcriptional start sites (TSS). f, Uniquely mapped IRF8 ChIP-Seq reads were evaluated for peak discovery by Model-based Analysis of ChIP-Seq (MACS) as described in Methods and peaks were annotated by the Homer software package using default annotation settings. Shown is the distribution of peak annotations within introns, intergenic regions, promoters, transcription termination sites (TTS), 3'-untranslated regions (3'UTR), exons, and 5'-untranslated regions (5'UTR). Numbers represent the percent of annotations in each category. g , Peaks identified in ( f ) were evaluated for de novo motif discovery using the Homer software package as described in Methods. Analysis was performed 3 separate times to identify the most significant motifs. Shown are the top 3 DNA motifs conserved in IRF8-bound loci along with the closest known motif and associated p -value (cumulative binomial distribution).
Figure Legend Snippet: IRF8 regulates the expression of Mycl1 in multiple dendritic cell lineages a , (left panels) BM cells and splenocytes from Mycl1 +/+ Irf8 +/+ , Mycl1 +/gfp Irf8 +/+ , Mycl1 +/+ Irf8 R294C , and Mycl1 +/gfp Irf8 R294C mice were stained for analysis. Shown are two-color histograms of GFP and CD11c expression. Numbers represent the percent of cells in the indicate gates. Data are representative of 3 independent experiments. (right panels) Ly6C + CD11b + BM monocytes were isolated by cell sorting from Mycl1 +/+ Irf8 +/+ (black line), Mycl1 +/+ Irf8 R294C (grey line), Mycl1 +/gfp Irf8 R294C (light green line), Mycl1 +/gfp Irf8 +/+ mice (dark green line) and differentiated for 4 days either with GM-CSF (left panel) or with GM-CSF and IL-4 (right panel). Shown are single-color histograms of GFP expression for pre-gated live CD11c + cells from the indicated genotypes. b , Splenocytes from WT (top panels) and Irf8 R294C (bottom panels) mice on the C57BL/6 genetic background were stained for analysis. Shown are two-color histograms of CD11c and SiglecH expression (left panels), and CD11b and CD205 expression (right panels), for cells pre-gated as either B220 + CD11b − or CD11c + MHCII + B220 − . pDCs were gated as CD11c + SiglecH + B220 + CD11b − . Numbers represent the percent of cells in the indicated gates. Data are representative of 2 independent experiments. c , Ly6C + BM monocytes were isolated by cell sorting from Mycl1 +/+ Irf8 +/+ , Mycl1 +/+ Irf8 R294C , Mycl1 +/gfp Irf8 R294C , and Mycl1 +/gfp Irf8 +/+ mice. Monocytes were cultured either with GM-CSF or with GM-CSF and IL-4 for 4 days and then stained for analysis. Shown are two-color histograms of MHCII and CD11c expression for differentiated monocytes. Numbers represent the percent of cells in the quadrant gate. Data are representative of 2 independent experiments. d , Phoenix-E cells were transfected with murine stem cell virus (MSCV) retroviral plasmids expressing the mutant human estrogen receptor (ER T2 ), murine Myc (c-MYC), murine Mycl1 (L-MYC), and a fusion between Mycl1 and the mutant human estrogen receptor (L-MYC-ER T2 ). Whole-cell extracts were prepared 2 days after transfection and Western blot analysis was performed. Shown are blots probed for L-MYC (left, α-L-MYC) and c-MYC (right, α-c-MYC) for the indicated transfections. Blots were stripped and re-probed for GFP (bottom, α-GFP). e , IRF8 DNA binding was assayed by ChIP-Seq. Shown is a histogram of normalized reads per bp per peak within a 4-kb window centered on transcriptional start sites (TSS). f, Uniquely mapped IRF8 ChIP-Seq reads were evaluated for peak discovery by Model-based Analysis of ChIP-Seq (MACS) as described in Methods and peaks were annotated by the Homer software package using default annotation settings. Shown is the distribution of peak annotations within introns, intergenic regions, promoters, transcription termination sites (TTS), 3'-untranslated regions (3'UTR), exons, and 5'-untranslated regions (5'UTR). Numbers represent the percent of annotations in each category. g , Peaks identified in ( f ) were evaluated for de novo motif discovery using the Homer software package as described in Methods. Analysis was performed 3 separate times to identify the most significant motifs. Shown are the top 3 DNA motifs conserved in IRF8-bound loci along with the closest known motif and associated p -value (cumulative binomial distribution).

Techniques Used: Expressing, Mouse Assay, Staining, Isolation, FACS, Cell Culture, Transfection, Mutagenesis, Western Blot, Binding Assay, Chromatin Immunoprecipitation, Magnetic Cell Separation, Software

Mycl1 regulates the homeostasis of CD8α + DCs and peripheral tissue CD103 + DCs a , Splenic DC subsets as gated in ( Extended Data Fig. 1e ) were quantitated from WT (black dots) and Mycl1 gfp/gfp (green dots) mice on the 129SvEv genetic background. Shown are graphs of total cell number per spleen for the indicated DC subsets (right panels) and of each DC subset as a percent of all splenocytes (left panels). Data are from 3 independent experiments (bar, SD: n=10 biological replicates, Student's t -test). b, c, d , Cells from the liver ( b ), lung ( c ), and small intestine ( d ) of WT and Mycl1 gfp/gfp mice on the C57BL/6 genetic background were stained for analysis. Shown are two-color histograms of 7AAD and CD45 expression, MHCII and CD11c expression, and CD103 and CD11b expression. Hematopoietic cells were gated as 7AAD − CD45 + (Live CD45 + ) and DCs were gated as MHCII + CD11c + . e , Cells from the lung, liver and small intestine of mixed BM chimeras described in ( Fig. 3a, c ) were analyzed for relative donor contribution. Shown is the contribution of CD45.2 + WT BM or CD45.2 + Mycl1 gpf/gfp BM to CD103 − CD11b + DCs in the indicated peripheral tissues as a ratio of peripheral blood neutrophils from the same animal. Data are representative of 3 independent experiments (bar, SD; n=5-10 mice, Mann-Whitney U test). f , Shown is relative Mycl1 mRNA expression determined by gene expression microarrays of lung-resident CD103 + CD11b − DCs and of migratory CD103 + CD11b − DCs from mediastinal lymph nodes (MLN) (bar, SD; n=3 biological replicate arrays, Student's t -test). g , Differences in microarray-based gene expression data between lung-resident CD103 + CD11b − DCs (Lung, red) and migratory CD103 + CD11b − DCs (Lymph node, blue) were analyzed for enrichment of a set of cell cycle genes (KEGG_Cell_Cycle) by Gene Set Enrichment Analysis (GSEA). Shown is a GSEA plot of running enrichment score (top), gene set member ranks (middle), and ranked list metric values (bottom) (signal-to-noise metric, n=3 biological replicate arrays per group). **, p
Figure Legend Snippet: Mycl1 regulates the homeostasis of CD8α + DCs and peripheral tissue CD103 + DCs a , Splenic DC subsets as gated in ( Extended Data Fig. 1e ) were quantitated from WT (black dots) and Mycl1 gfp/gfp (green dots) mice on the 129SvEv genetic background. Shown are graphs of total cell number per spleen for the indicated DC subsets (right panels) and of each DC subset as a percent of all splenocytes (left panels). Data are from 3 independent experiments (bar, SD: n=10 biological replicates, Student's t -test). b, c, d , Cells from the liver ( b ), lung ( c ), and small intestine ( d ) of WT and Mycl1 gfp/gfp mice on the C57BL/6 genetic background were stained for analysis. Shown are two-color histograms of 7AAD and CD45 expression, MHCII and CD11c expression, and CD103 and CD11b expression. Hematopoietic cells were gated as 7AAD − CD45 + (Live CD45 + ) and DCs were gated as MHCII + CD11c + . e , Cells from the lung, liver and small intestine of mixed BM chimeras described in ( Fig. 3a, c ) were analyzed for relative donor contribution. Shown is the contribution of CD45.2 + WT BM or CD45.2 + Mycl1 gpf/gfp BM to CD103 − CD11b + DCs in the indicated peripheral tissues as a ratio of peripheral blood neutrophils from the same animal. Data are representative of 3 independent experiments (bar, SD; n=5-10 mice, Mann-Whitney U test). f , Shown is relative Mycl1 mRNA expression determined by gene expression microarrays of lung-resident CD103 + CD11b − DCs and of migratory CD103 + CD11b − DCs from mediastinal lymph nodes (MLN) (bar, SD; n=3 biological replicate arrays, Student's t -test). g , Differences in microarray-based gene expression data between lung-resident CD103 + CD11b − DCs (Lung, red) and migratory CD103 + CD11b − DCs (Lymph node, blue) were analyzed for enrichment of a set of cell cycle genes (KEGG_Cell_Cycle) by Gene Set Enrichment Analysis (GSEA). Shown is a GSEA plot of running enrichment score (top), gene set member ranks (middle), and ranked list metric values (bottom) (signal-to-noise metric, n=3 biological replicate arrays per group). **, p

Techniques Used: Mouse Assay, Staining, Expressing, MANN-WHITNEY, Microarray

18) Product Images from "Differential Type I Interferon-dependent Transgene Silencing of Helper-dependent Adenoviral vs. Adeno-associated Viral Vectors In Vivo"

Article Title: Differential Type I Interferon-dependent Transgene Silencing of Helper-dependent Adenoviral vs. Adeno-associated Viral Vectors In Vivo

Journal: Molecular Therapy

doi: 10.1038/mt.2012.277

Type I interferon (IFN) signaling suppresses transgene expression of Helper-dependent adenoviral vector (HDAd) at transcriptional level in livers of mice. Wild-type (WT) C57Bl/6 and IFNαR −/− mice were injected with 5 ×
Figure Legend Snippet: Type I interferon (IFN) signaling suppresses transgene expression of Helper-dependent adenoviral vector (HDAd) at transcriptional level in livers of mice. Wild-type (WT) C57Bl/6 and IFNαR −/− mice were injected with 5 ×

Techniques Used: Expressing, Plasmid Preparation, Mouse Assay, Injection

Promyelocytic leukemia protein (PML) mRNA is induced dependently on type I interferon (IFN) signaling and associated with Helper-dependent adenoviral vector (HDAd) DNA in livers of wild-type (WT) mice. WT C57Bl/6 mice were injected with 5 × 10
Figure Legend Snippet: Promyelocytic leukemia protein (PML) mRNA is induced dependently on type I interferon (IFN) signaling and associated with Helper-dependent adenoviral vector (HDAd) DNA in livers of wild-type (WT) mice. WT C57Bl/6 mice were injected with 5 × 10

Techniques Used: Plasmid Preparation, Mouse Assay, Injection

Contribution of type I interferon (IFN) signaling to transgene expression of Helper-dependent adenoviral vectors (HDAds) or scAAV2 at long-time points . ( a ) Wild-type (WT) C57Bl/6 and IFNαR −/− mice were injected with 5 ×
Figure Legend Snippet: Contribution of type I interferon (IFN) signaling to transgene expression of Helper-dependent adenoviral vectors (HDAds) or scAAV2 at long-time points . ( a ) Wild-type (WT) C57Bl/6 and IFNαR −/− mice were injected with 5 ×

Techniques Used: Expressing, Mouse Assay, Injection

Type I interferon (IFN) mRNA is induced in livers at 1 hour post-injection of HDAd LacZ and amplifies cytokine expression in livers and blood of mice. Wild-type (WT) C57Bl/6 mice were injected with 5 × 10 11 viral particle (Vp)/kg of HDAd LacZ into
Figure Legend Snippet: Type I interferon (IFN) mRNA is induced in livers at 1 hour post-injection of HDAd LacZ and amplifies cytokine expression in livers and blood of mice. Wild-type (WT) C57Bl/6 mice were injected with 5 × 10 11 viral particle (Vp)/kg of HDAd LacZ into

Techniques Used: Injection, Expressing, Mouse Assay

ScAAV2/8 attenuates type I interferon (IFN) signaling dependent transgene silencing at 24 hours post-injection. ( a ) Wild-type (WT) C57Bl/6 and IFNαR −/− mice were injected with 5 × 10 11 Vg/kg of scAAV2/8 EGFP into tail veins.
Figure Legend Snippet: ScAAV2/8 attenuates type I interferon (IFN) signaling dependent transgene silencing at 24 hours post-injection. ( a ) Wild-type (WT) C57Bl/6 and IFNαR −/− mice were injected with 5 × 10 11 Vg/kg of scAAV2/8 EGFP into tail veins.

Techniques Used: Injection, Mouse Assay

19) Product Images from "Dengue and Zika Virus Cross-Reactive Human Monoclonal Antibodies Protect against Spondweni Virus Infection and Pathogenesis in Mice"

Article Title: Dengue and Zika Virus Cross-Reactive Human Monoclonal Antibodies Protect against Spondweni Virus Infection and Pathogenesis in Mice

Journal: Cell reports

doi: 10.1016/j.celrep.2019.01.052

Clinical Consequences of SPOV Infection in Mice (A–D) Survival and weight loss analysis. 8-week-old C57BL/6 male micewere pretreated at day −1 with no antibody or a single dose of anti-Ifnar1 mAb (0.5, 1, or 2 mg) and then mock-infected or inoculated with 10 6 FFU of SPOV-SA Ar94 (A and B) or 10 5 FFU of SPOV-Chuku (C and D) and followed for survival (A and C) or weight change (B and D). Survival analysis was compared to the uninfected mice (log rank-test with Bonferroni post-test: n.s., not significant; **p
Figure Legend Snippet: Clinical Consequences of SPOV Infection in Mice (A–D) Survival and weight loss analysis. 8-week-old C57BL/6 male micewere pretreated at day −1 with no antibody or a single dose of anti-Ifnar1 mAb (0.5, 1, or 2 mg) and then mock-infected or inoculated with 10 6 FFU of SPOV-SA Ar94 (A and B) or 10 5 FFU of SPOV-Chuku (C and D) and followed for survival (A and C) or weight change (B and D). Survival analysis was compared to the uninfected mice (log rank-test with Bonferroni post-test: n.s., not significant; **p

Techniques Used: Infection, Mouse Assay

SPOV Infection at the Maternal-Fetal Interface (A–C) 8- to 10-week-old pregnant C57BL/6 dams were treated on E5.5 with no mAb or anti-Ifnar1 mAb (0.5 or 1 mg) and then inoculated via subcutaneous route with 10 6 FFU of SPOV-SA Ar94 on E6.5. At E13.5, maternal (A) and fetal (B and C) tissues were processed for SPOV RNA (A and B) or infectious virus (C) by qRT-PCR and plaque assays, respectively. Levels of viral RNA or infectious virus were compared to those obtained from mice treated with no mAb (one-way ANOVA with Dunnett’s post-test: **p
Figure Legend Snippet: SPOV Infection at the Maternal-Fetal Interface (A–C) 8- to 10-week-old pregnant C57BL/6 dams were treated on E5.5 with no mAb or anti-Ifnar1 mAb (0.5 or 1 mg) and then inoculated via subcutaneous route with 10 6 FFU of SPOV-SA Ar94 on E6.5. At E13.5, maternal (A) and fetal (B and C) tissues were processed for SPOV RNA (A and B) or infectious virus (C) by qRT-PCR and plaque assays, respectively. Levels of viral RNA or infectious virus were compared to those obtained from mice treated with no mAb (one-way ANOVA with Dunnett’s post-test: **p

Techniques Used: Infection, Quantitative RT-PCR, Mouse Assay

Immune Responses after SPOV or ZIKV Infection 8-week-old C57BL/6 male mice were uninfected or pretreated at day −1 with a single 0.5-mg dose of anti-Ifnar1 mAb and then inoculated with 10 6 FFU of SPOV-SA Ar94 or ZIKV Dakar 41525. (A) At 8 dpi, spleens were harvested and processed by flow cytometry. The total number of indicated cell populations (CD19 + , CD4 + , CD44 + CD4 + , CD8 + , and granzyme B + CD8 + ) is shown. Cells also were incubated with an immunodominant CD8 + T cell peptide and stained intracellularly for IFN-γ. Cell numbers were compared to those obtained from uninfected mice (one-way ANOVA with Dunnett’s post-test: **p
Figure Legend Snippet: Immune Responses after SPOV or ZIKV Infection 8-week-old C57BL/6 male mice were uninfected or pretreated at day −1 with a single 0.5-mg dose of anti-Ifnar1 mAb and then inoculated with 10 6 FFU of SPOV-SA Ar94 or ZIKV Dakar 41525. (A) At 8 dpi, spleens were harvested and processed by flow cytometry. The total number of indicated cell populations (CD19 + , CD4 + , CD44 + CD4 + , CD8 + , and granzyme B + CD8 + ) is shown. Cells also were incubated with an immunodominant CD8 + T cell peptide and stained intracellularly for IFN-γ. Cell numbers were compared to those obtained from uninfected mice (one-way ANOVA with Dunnett’s post-test: **p

Techniques Used: Infection, Mouse Assay, Flow Cytometry, Cytometry, Incubation, Staining

Viral Burden in SPOV-Infected Mice (A–F) 8-week-old C57BL/6 male mice were pretreated at day −1 with no antibody or a single dose of anti-Ifnar1 mAb (0.5 or 1 mg) and then inoculated with 10 6 FFU of SPOV-SA Ar94 (A, C, and E) or 10 5 FFU of SPOV-Chuku (B, D, and F). Serum and tissues (brain, spleen, testis, kidney, ileum, and eye) were harvested at days 7 (A and B), 14 (C and D), or 21 (E and F) after infection, and SPOV RNA levels were measured by qRT-PCR. Viral RNA levels were compared to tissues from the no anti-Ifnar1 mAb-treated mice (one-way ANOVA with Dunnett’s post-test: ***p
Figure Legend Snippet: Viral Burden in SPOV-Infected Mice (A–F) 8-week-old C57BL/6 male mice were pretreated at day −1 with no antibody or a single dose of anti-Ifnar1 mAb (0.5 or 1 mg) and then inoculated with 10 6 FFU of SPOV-SA Ar94 (A, C, and E) or 10 5 FFU of SPOV-Chuku (B, D, and F). Serum and tissues (brain, spleen, testis, kidney, ileum, and eye) were harvested at days 7 (A and B), 14 (C and D), or 21 (E and F) after infection, and SPOV RNA levels were measured by qRT-PCR. Viral RNA levels were compared to tissues from the no anti-Ifnar1 mAb-treated mice (one-way ANOVA with Dunnett’s post-test: ***p

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR

Neutralization and Protection against SPOV Infection by Cross-Reactive Anti-DENV or Anti-ZIKV mAbs (A) Neutralization of SPOV-SA Ar94 by anti-DENV (EDE1-B10, EDE1-A9, and EDE1-C4) or anti-ZIKV (ZIKV-A7, ZIKV-C10, or ZIKV-117) mAbs as determined by focus reduction neutralization test in Vero cells. LALA variants of EDE1-B10 and ZIKV-117 lacking the ability to bind FcγR also were tested. The data are representative of three experiments, each performed in triplicate. (B) Binding of parental and LALA variants of hu-CHK-152, ZIKV-117, and EDE1-B10 to human FcγRI (CD64) by ELISA. (C–F) Prophylaxis and post-exposure treatment studies. 8-week-old C57BL/6 male mice were treated with anti-Ifnar1 mAb at day −1 followed by subcutaneous infection with 10 6 FFU of SPOV-SA Ar94. Mice also were treated at day −1 (prophylaxis) or day +1 (post-exposure) with isotype-control mAb (hu-CHK-152) or with a single 100-μg dose of EDE1-B10, EDE1-A9, EDE1-C4, ZIKV-A7, ZIKV-C10, or ZIKV-117. Survival (C and E) and weight (D and F) data were from two experiments (n = 9 or 10 per group; survival: log rank test with Bonferroni post-test; weight: Kruskal-Wallis ANOVA with Dunnett’s post-test). The bracketed bar indicates statistical significance (****p
Figure Legend Snippet: Neutralization and Protection against SPOV Infection by Cross-Reactive Anti-DENV or Anti-ZIKV mAbs (A) Neutralization of SPOV-SA Ar94 by anti-DENV (EDE1-B10, EDE1-A9, and EDE1-C4) or anti-ZIKV (ZIKV-A7, ZIKV-C10, or ZIKV-117) mAbs as determined by focus reduction neutralization test in Vero cells. LALA variants of EDE1-B10 and ZIKV-117 lacking the ability to bind FcγR also were tested. The data are representative of three experiments, each performed in triplicate. (B) Binding of parental and LALA variants of hu-CHK-152, ZIKV-117, and EDE1-B10 to human FcγRI (CD64) by ELISA. (C–F) Prophylaxis and post-exposure treatment studies. 8-week-old C57BL/6 male mice were treated with anti-Ifnar1 mAb at day −1 followed by subcutaneous infection with 10 6 FFU of SPOV-SA Ar94. Mice also were treated at day −1 (prophylaxis) or day +1 (post-exposure) with isotype-control mAb (hu-CHK-152) or with a single 100-μg dose of EDE1-B10, EDE1-A9, EDE1-C4, ZIKV-A7, ZIKV-C10, or ZIKV-117. Survival (C and E) and weight (D and F) data were from two experiments (n = 9 or 10 per group; survival: log rank test with Bonferroni post-test; weight: Kruskal-Wallis ANOVA with Dunnett’s post-test). The bracketed bar indicates statistical significance (****p

Techniques Used: Neutralization, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Mouse Assay

20) Product Images from "CD36 Upregulation Mediated by Intranasal LV-NRF2 Treatment Mitigates Hypoxia-Induced Progression of Alzheimer's-Like Pathogenesis"

Article Title: CD36 Upregulation Mediated by Intranasal LV-NRF2 Treatment Mitigates Hypoxia-Induced Progression of Alzheimer's-Like Pathogenesis

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2014.5845

Representative images of Aβ plaques in APP/PS1 and age-matched C57BL/6 mice brain. Aβ plaques were examined with anti-Aβ antibody. Aβ-positive plaques were significantly detectable in the cortex and hippocampus of APP/PS1
Figure Legend Snippet: Representative images of Aβ plaques in APP/PS1 and age-matched C57BL/6 mice brain. Aβ plaques were examined with anti-Aβ antibody. Aβ-positive plaques were significantly detectable in the cortex and hippocampus of APP/PS1

Techniques Used: Mouse Assay

Hypoxia treatment impairs the spatial learning and memory in the aging APP/PS1 mice. Two-, six-, and 12-month-old double APP/PS1 transgenic (Tg) and age-matched wild-type (WT) C57BL/6 mice were treated with hypoxia intervention (Tg+Hypo, WT+Hypo) or control
Figure Legend Snippet: Hypoxia treatment impairs the spatial learning and memory in the aging APP/PS1 mice. Two-, six-, and 12-month-old double APP/PS1 transgenic (Tg) and age-matched wild-type (WT) C57BL/6 mice were treated with hypoxia intervention (Tg+Hypo, WT+Hypo) or control

Techniques Used: Mouse Assay, Transgenic Assay

Hypoxia treatment markedly increases β-amyloid burden in the aging APP/PS1 mouse brain. Levels of mouse soluble Aβ1-40 (A) and Aβ1-42 (B) were determined by ELISA in the APP/PS1 transgenic (Tg) and age-matched WT C57BL/6 mouse
Figure Legend Snippet: Hypoxia treatment markedly increases β-amyloid burden in the aging APP/PS1 mouse brain. Levels of mouse soluble Aβ1-40 (A) and Aβ1-42 (B) were determined by ELISA in the APP/PS1 transgenic (Tg) and age-matched WT C57BL/6 mouse

Techniques Used: Enzyme-linked Immunosorbent Assay, Transgenic Assay

21) Product Images from "Platelet TGF-?1 contributions to plasma TGF-?1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload"

Article Title: Platelet TGF-?1 contributions to plasma TGF-?1, cardiac fibrosis, and systolic dysfunction in a mouse model of pressure overload

Journal: Blood

doi: 10.1182/blood-2011-09-377648

Induction of profound thrombocytopenia by mAb 1B5 injection reduces plasma TGF-β1 levels in mice. WT C57BL/6 mice were injected intraperitoneally with the indicated amount of hamster mAb 1B5 against mouse αIIbβ3. (A) Platelet counts
Figure Legend Snippet: Induction of profound thrombocytopenia by mAb 1B5 injection reduces plasma TGF-β1 levels in mice. WT C57BL/6 mice were injected intraperitoneally with the indicated amount of hamster mAb 1B5 against mouse αIIbβ3. (A) Platelet counts

Techniques Used: Injection, Mouse Assay

22) Product Images from "Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer"

Article Title: Paneth cell marker expression in intestinal villi and colon crypts characterizes dietary induced risk for mouse sporadic intestinal cancer

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1017668108

Detection of Wnt signaling by assay of β-galactosidase activity (blue) in frozen sections of the mucosa of C57BL/6 mice that harbor a lacZ transgene driven by a minimal fos promoter under regulation of three tandem Tcf4 binding sites. In this case, mice were fed the three diets from weaning for 3 mo.
Figure Legend Snippet: Detection of Wnt signaling by assay of β-galactosidase activity (blue) in frozen sections of the mucosa of C57BL/6 mice that harbor a lacZ transgene driven by a minimal fos promoter under regulation of three tandem Tcf4 binding sites. In this case, mice were fed the three diets from weaning for 3 mo.

Techniques Used: Activity Assay, Mouse Assay, Binding Assay

23) Product Images from "Hemorrhagic shock primes for lung vascular endothelial cell pyroptosis: role in pulmonary inflammation following LPS"

Article Title: Hemorrhagic shock primes for lung vascular endothelial cell pyroptosis: role in pulmonary inflammation following LPS

Journal: Cell Death & Disease

doi: 10.1038/cddis.2016.274

HS primes for lung endothelial cell pyroptosis in response to LPS through HMGB1-RAGE signaling. ( a ) WT (C57BL/6) mice, TLR4 −/− mice, RAGE −/− mice, and Nlrp3 −/− mice were subjected to HS (HS) or sham operation (Sham) followed by LPS or saline (SAL) i.t. at 2 h after resuscitation ( n =6 per group). Some WT mice received anti-HMGB1 Ab (2 mg/kg BW) by i.p. injection 30 min before HS or sham operation ( n =6 per group). Lung tissue were harvested 24 h after LPS or SAL i.t. and the histological slides were stained with Cell Death Reagent-TMR (red), Alexa Fluor 488-labeled caspase-1 FLICA (green), E-selectin (white), and Hoechst (blue). Fluorescent images were obtained by confocal microscopy (original magnification × 600, higher magnification images for the selected area are shown in the respective lower right insets). Quadruple-stained cells were considered positive for pyroptotic EC. ( b ) The average number of pyroptotic EC of five random fields was counted for analysis. Data are presented as mean and S.E.M. * P
Figure Legend Snippet: HS primes for lung endothelial cell pyroptosis in response to LPS through HMGB1-RAGE signaling. ( a ) WT (C57BL/6) mice, TLR4 −/− mice, RAGE −/− mice, and Nlrp3 −/− mice were subjected to HS (HS) or sham operation (Sham) followed by LPS or saline (SAL) i.t. at 2 h after resuscitation ( n =6 per group). Some WT mice received anti-HMGB1 Ab (2 mg/kg BW) by i.p. injection 30 min before HS or sham operation ( n =6 per group). Lung tissue were harvested 24 h after LPS or SAL i.t. and the histological slides were stained with Cell Death Reagent-TMR (red), Alexa Fluor 488-labeled caspase-1 FLICA (green), E-selectin (white), and Hoechst (blue). Fluorescent images were obtained by confocal microscopy (original magnification × 600, higher magnification images for the selected area are shown in the respective lower right insets). Quadruple-stained cells were considered positive for pyroptotic EC. ( b ) The average number of pyroptotic EC of five random fields was counted for analysis. Data are presented as mean and S.E.M. * P

Techniques Used: Mouse Assay, Injection, Staining, Labeling, Confocal Microscopy

24) Product Images from "Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema"

Article Title: Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1005574107

LC3B regulates CS-induced autophagy, apoptosis, and emphysematous airspace enlargement in vivo. WT C57BL/6, LC3B +/+ , or LC3B −/− mice were exposed to chronic CS exposure or air for 3 mo (C57BL/6, n = 9 for air, n = 8 for CS; LC3B +/+ , n
Figure Legend Snippet: LC3B regulates CS-induced autophagy, apoptosis, and emphysematous airspace enlargement in vivo. WT C57BL/6, LC3B +/+ , or LC3B −/− mice were exposed to chronic CS exposure or air for 3 mo (C57BL/6, n = 9 for air, n = 8 for CS; LC3B +/+ , n

Techniques Used: In Vivo, Mouse Assay

Cav-1 regulates CS-induced autophagy and apoptosis in vivo. WT C57BL/6 or Cav-1 −/− mice were exposed to chronic CS exposure for 3 mo (each group, n = 5). ( A ) EM images were scored for number of AVs. The data are represented as AVs per
Figure Legend Snippet: Cav-1 regulates CS-induced autophagy and apoptosis in vivo. WT C57BL/6 or Cav-1 −/− mice were exposed to chronic CS exposure for 3 mo (each group, n = 5). ( A ) EM images were scored for number of AVs. The data are represented as AVs per

Techniques Used: In Vivo, Mouse Assay

25) Product Images from "T-bet Knockout Prevents Helicobacter felis-Induced Gastric Cancer"

Article Title: T-bet Knockout Prevents Helicobacter felis-Induced Gastric Cancer

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.0900511

Histology of uninfected WT and T-bet mice are similar. H E staining of WT C57BL/6 mouse ( A , ×4; B , ×40) and T-bet KO mouse ( C , ×4; D , ×40). Parietal cells (arrows) are large cells with pale pink cytoplasm and centrally
Figure Legend Snippet: Histology of uninfected WT and T-bet mice are similar. H E staining of WT C57BL/6 mouse ( A , ×4; B , ×40) and T-bet KO mouse ( C , ×4; D , ×40). Parietal cells (arrows) are large cells with pale pink cytoplasm and centrally

Techniques Used: Mouse Assay, Staining

26) Product Images from "A novel IL-17 signaling pathway controlling keratinocyte proliferation and tumorigenesis via the TRAF4–ERK5 axis"

Article Title: A novel IL-17 signaling pathway controlling keratinocyte proliferation and tumorigenesis via the TRAF4–ERK5 axis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20150204

The MEKK3–ERK5 axis is critical for IL-17–induced epidermal keratinocyte proliferation. (A) Western blot analysis of epidermal lysates from ears of WT and IL-17RC −/− mice injected with IL-17A for the indicated times. (B) H E staining of ear sections of C57BL/6 WT mice injected with PBS, IL-17A in the presence or absence of ERK5 siRNA (left), or MEK5 inhibitor Bix 02189 (right). Graphs represent mean epidermal thickness (a.u.) ± SEM. ***, P
Figure Legend Snippet: The MEKK3–ERK5 axis is critical for IL-17–induced epidermal keratinocyte proliferation. (A) Western blot analysis of epidermal lysates from ears of WT and IL-17RC −/− mice injected with IL-17A for the indicated times. (B) H E staining of ear sections of C57BL/6 WT mice injected with PBS, IL-17A in the presence or absence of ERK5 siRNA (left), or MEK5 inhibitor Bix 02189 (right). Graphs represent mean epidermal thickness (a.u.) ± SEM. ***, P

Techniques Used: Western Blot, Mouse Assay, Injection, Staining

27) Product Images from "MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation"

Article Title: MHCII glycosylation modulates Bacteroides fragilis carbohydrate antigen presentation

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20100508

Native complex N-glycans are required for PSA-driven T cell activation in vitro. (A) BMDCs were cultured in the presence or absence of CS (red) and KF (blue) for 3 d, and then analyzed by PHA-L, PHA-E, WGA, and Con A lectin flow cytometry to detect N-glycans. Representative histograms and n = 4 MFI analyses are shown. (B) BMDCs were cultured for 8 d, probed with CD11c mAb, and analyzed by flow cytometry to verify proper DC cell differentiation. Representative histogram is shown. (C) BMDCs were incubated with nonfluorescent intact DQ-OVA protein for 24 h to allow uptake and processing. Cells were then analyzed for fluorescence, which is indicative of endocytosis and cleavage to peptides. Representative histograms are shown. n = 4 for MFI analyses. (D) BMDC maturation upon exposure to antigen was measured by surface staining of MHCII 24 h after stimulation with OVA peptide and LPS (O+L), LPS alone, or PSA. Representative histograms shown. (E) BMDCs grown with and without CS and KF were pulsed with either DQ-OVA (OVA), OVAp+LPS (O+L), or LPS alone for 24 h, after which untreated fresh naive CD4 + T cells isolated from OT-II animals were added. n = 3 per condition, per antigen. (F) As with OVAp, BMDCs were grown with CS and KF and pulsed with PSA, Sp1, or B. fragilis capsule (Caps) for 24 h. Next, naive WT C57BL/6 CD4 + T cells were added to allow activation to occur. n = 3 per condition. (G) TLR2 activation by PSA, a necessary step in GlyAg-mediated T cell activation ( Wang et al., 2006 ), and a control agonist Pam3Cysk4 (P3C) was measured in untreated (UNT), CS-treated, or KF-treated TLR2 + HEK293 cells. Bars represent background-subtracted values. n = 3 per condition. All bar graphs show the mean ± SEM.
Figure Legend Snippet: Native complex N-glycans are required for PSA-driven T cell activation in vitro. (A) BMDCs were cultured in the presence or absence of CS (red) and KF (blue) for 3 d, and then analyzed by PHA-L, PHA-E, WGA, and Con A lectin flow cytometry to detect N-glycans. Representative histograms and n = 4 MFI analyses are shown. (B) BMDCs were cultured for 8 d, probed with CD11c mAb, and analyzed by flow cytometry to verify proper DC cell differentiation. Representative histogram is shown. (C) BMDCs were incubated with nonfluorescent intact DQ-OVA protein for 24 h to allow uptake and processing. Cells were then analyzed for fluorescence, which is indicative of endocytosis and cleavage to peptides. Representative histograms are shown. n = 4 for MFI analyses. (D) BMDC maturation upon exposure to antigen was measured by surface staining of MHCII 24 h after stimulation with OVA peptide and LPS (O+L), LPS alone, or PSA. Representative histograms shown. (E) BMDCs grown with and without CS and KF were pulsed with either DQ-OVA (OVA), OVAp+LPS (O+L), or LPS alone for 24 h, after which untreated fresh naive CD4 + T cells isolated from OT-II animals were added. n = 3 per condition, per antigen. (F) As with OVAp, BMDCs were grown with CS and KF and pulsed with PSA, Sp1, or B. fragilis capsule (Caps) for 24 h. Next, naive WT C57BL/6 CD4 + T cells were added to allow activation to occur. n = 3 per condition. (G) TLR2 activation by PSA, a necessary step in GlyAg-mediated T cell activation ( Wang et al., 2006 ), and a control agonist Pam3Cysk4 (P3C) was measured in untreated (UNT), CS-treated, or KF-treated TLR2 + HEK293 cells. Bars represent background-subtracted values. n = 3 per condition. All bar graphs show the mean ± SEM.

Techniques Used: Activation Assay, In Vitro, Cell Culture, Whole Genome Amplification, Flow Cytometry, Cytometry, Cell Differentiation, Incubation, Fluorescence, Staining, Isolation

Native complex N-glycans are required for PSA-driven immune responses in vivo. (A) WT C57BL/6 animals ( n = 8) were treated daily for 1, 2, or 3 d with 250 µg of KF, and then sacrificed to harvest splenocytes. Cells were stained for native complex N-glycans using PHA-L. Representative histogram shown. (B) WT mice ( n = 3 per group) were treated with KF or PBS for 3 d, as in A. Animals were then sensitized with 100 µg PSA. Splenocytes were harvested 6 d later, stained for IFN-γ and CD4, and analyzed by flow cytometry. (C) KF- or PBS-pretreated mice ( n = 6) were sensitized with 100 µg OVA and sterile cecal content adjuvant. 4 d later, CD4 + T cells were isolated and restimulated ex vivo with normally glycosylated APCs and OVA ( n = 6). (D) Animals ( n = 6) were treated as in C, only with 100 µg of PSA rather than OVA. The PSA-specific recall ( n = 18) was measured after ex vivo restimulation with normally glycosylated APCs and PSA. (E) Animals ( n = 6) were treated as in D, only with the additional adoptive transfer of 5 × 10 6 fresh normally glycosylated APCs at the time of PSA injection ( n = 12). For panels C–E, data were normalized to the antigen-sensitized animals without KF. All bar graphs show the mean ± SEM.
Figure Legend Snippet: Native complex N-glycans are required for PSA-driven immune responses in vivo. (A) WT C57BL/6 animals ( n = 8) were treated daily for 1, 2, or 3 d with 250 µg of KF, and then sacrificed to harvest splenocytes. Cells were stained for native complex N-glycans using PHA-L. Representative histogram shown. (B) WT mice ( n = 3 per group) were treated with KF or PBS for 3 d, as in A. Animals were then sensitized with 100 µg PSA. Splenocytes were harvested 6 d later, stained for IFN-γ and CD4, and analyzed by flow cytometry. (C) KF- or PBS-pretreated mice ( n = 6) were sensitized with 100 µg OVA and sterile cecal content adjuvant. 4 d later, CD4 + T cells were isolated and restimulated ex vivo with normally glycosylated APCs and OVA ( n = 6). (D) Animals ( n = 6) were treated as in C, only with 100 µg of PSA rather than OVA. The PSA-specific recall ( n = 18) was measured after ex vivo restimulation with normally glycosylated APCs and PSA. (E) Animals ( n = 6) were treated as in D, only with the additional adoptive transfer of 5 × 10 6 fresh normally glycosylated APCs at the time of PSA injection ( n = 12). For panels C–E, data were normalized to the antigen-sensitized animals without KF. All bar graphs show the mean ± SEM.

Techniques Used: In Vivo, Staining, Mouse Assay, Flow Cytometry, Cytometry, Isolation, Ex Vivo, Adoptive Transfer Assay, Injection

28) Product Images from "Integrin αv β8–expressing tumor cells evade host immunity by regulating TGF- β activation in immune cells"

Article Title: Integrin αv β8–expressing tumor cells evade host immunity by regulating TGF- β activation in immune cells

Journal: JCI Insight

doi: 10.1172/jci.insight.122591

MC38 colon carcinoma tumor growth in vivo is reduced by anti-αvβ8 and eliminated in the majority of mice by combination with anti–PD-1. ( A . ( B ) MC38 cells express cell-surface PD-L1. Cells were stained with anti–PD-L1 (light gray). As a positive control cells were stimulated with IFN-γ (dark gray) (10 ng/ml) for 24 hours (hr). Shown is a representative histogram overlay of PD-L1 expression of IFN-γ–stimulated or nonstimulated compared with control (a representative experiment of 4 is shown). ( C ) MC38 cells support αvβ8-mediated TGF-β activation. MC38 cells were cocultured with TGF-β reporter cells in the presence or absence of C6D4, isotype control, or pan–TGF-β neutralizing antibody (1D11). Dotted line indicates basal level of TGF-β activation in MC38 cells. ( D–I ) MC38 colon carcinoma cells (5 × 10 5 ) were s.c. implanted into the flank of C57BL/6 mice. After tumors became palpable (7 days after inoculation), isotype control (anti-SV5 and 2A3), anti-β8 (C6D4), anti–PD-1 (RMP1-14), or both in combination were injected on days 0, 3, 6 (10 mg/kg i.p.), and RMP1-14 was added alone on day 9 (10 mg/kg i.p.). Spider plots of tumor growth of each treatment arm from mice treated with isotype control ( D ) SV5 and 2A3 (mouse and rat IgG2a, respectively) ( n = 10) and neutralizing antibodies to ( E ) αvβ8 (C6D4) ( n = 10), ( G ) PD-1 (RMP1-14) ( n = 9), or ( H ) αvβ8 and PD-1 (C6D4 and RMP1-14) ( n = 10). ( F ) Average tumor volumes from D , E , G , and H 15 days after tumor cell injection and 7 days after antibody administration is shown. ( I ) Kaplan-Meier survival plots. In legends F and I , ANOVA with Tukey’s post-hoc test of day 7 volume, or day 70 survival data, respectively, is shown. * P
Figure Legend Snippet: MC38 colon carcinoma tumor growth in vivo is reduced by anti-αvβ8 and eliminated in the majority of mice by combination with anti–PD-1. ( A . ( B ) MC38 cells express cell-surface PD-L1. Cells were stained with anti–PD-L1 (light gray). As a positive control cells were stimulated with IFN-γ (dark gray) (10 ng/ml) for 24 hours (hr). Shown is a representative histogram overlay of PD-L1 expression of IFN-γ–stimulated or nonstimulated compared with control (a representative experiment of 4 is shown). ( C ) MC38 cells support αvβ8-mediated TGF-β activation. MC38 cells were cocultured with TGF-β reporter cells in the presence or absence of C6D4, isotype control, or pan–TGF-β neutralizing antibody (1D11). Dotted line indicates basal level of TGF-β activation in MC38 cells. ( D–I ) MC38 colon carcinoma cells (5 × 10 5 ) were s.c. implanted into the flank of C57BL/6 mice. After tumors became palpable (7 days after inoculation), isotype control (anti-SV5 and 2A3), anti-β8 (C6D4), anti–PD-1 (RMP1-14), or both in combination were injected on days 0, 3, 6 (10 mg/kg i.p.), and RMP1-14 was added alone on day 9 (10 mg/kg i.p.). Spider plots of tumor growth of each treatment arm from mice treated with isotype control ( D ) SV5 and 2A3 (mouse and rat IgG2a, respectively) ( n = 10) and neutralizing antibodies to ( E ) αvβ8 (C6D4) ( n = 10), ( G ) PD-1 (RMP1-14) ( n = 9), or ( H ) αvβ8 and PD-1 (C6D4 and RMP1-14) ( n = 10). ( F ) Average tumor volumes from D , E , G , and H 15 days after tumor cell injection and 7 days after antibody administration is shown. ( I ) Kaplan-Meier survival plots. In legends F and I , ANOVA with Tukey’s post-hoc test of day 7 volume, or day 70 survival data, respectively, is shown. * P

Techniques Used: In Vivo, Mouse Assay, Staining, Positive Control, Expressing, Activation Assay, Injection

29) Product Images from "Reciprocal regulation of Th2 and Th17 cells by PAD2-mediated citrullination"

Article Title: Reciprocal regulation of Th2 and Th17 cells by PAD2-mediated citrullination

Journal: JCI Insight

doi: 10.1172/jci.insight.129687

Recapitulating the effects of Cl-am by inhibiting PAD2 but not PAD4. ( A ) The transcript levels of indicated PADs in Th cells ( n = 3) and macrophages ( n = 2) of C57BL/6 mice were measured with real-time PCR and normalized against the levels of actin. ( B ) The levels of PAD2 proteins in indicated C57BL/6 Th cells and PAD2-KO Th2 cells were examined with Western blotting using indicated antibodies. ( C ) At indicated time points during the differentiation of WT Th2 and Th17 cells, the level of PAD2 protein was examined with Western blotting. A parallel gel was probed with anti-tubulin for loading control. ( D and E ) WT Th cells were stimulated with anti-CD3, anti-CD28, and/or IL-2 in the absence ( D ) or presence ( E ) of cyclosporine A (CsA) or sotrastaurin (Sot) for 2 days. The level of PAD2 proteins was examined with Western blotting. Representative Western blots from 2 experiments are shown in B – D . ( F ) C57BL/6 effector Th cells were generated in the presence of AFM30a (left panel) or GSK199 (right panel) at indicated concentrations. The expression of indicated cytokines by the Th cells after restimulation with anti-CD3 is shown ( n = 3–4, 1-way ANOVA) . ( G – J ) Th cells obtained from WT DBA/1J ( G – J , n = 3–4), PAD2-KO ( G , I , and J , n = 3–5), or PAD4-KO ( H , n = 2) mice were differentiated into Th1, Th2, and Th17 cells for 5 days. The production of indicated cytokines after restimulation with anti-CD3 was measured with ELISA or real-time PCR ( G and H , 2-tailed Student’s t test for G ). Data from the same experiments are connected with lines in H . Whole cell extract from WT DBA/1J or PAD2-KO Th cells were examined with Western blotting using anti-citH3 ( I ). A parallel gel was probed with anti-H3 for loading controls. The normalized density of cit-H3 of indicated cells is shown in the dot blot of I . The transcript levels of PADs in restimulated WT DBA/1J, and PAD2-KO Th2 cells were quantified with real-time PCR and shown in J ( n = 3–4).
Figure Legend Snippet: Recapitulating the effects of Cl-am by inhibiting PAD2 but not PAD4. ( A ) The transcript levels of indicated PADs in Th cells ( n = 3) and macrophages ( n = 2) of C57BL/6 mice were measured with real-time PCR and normalized against the levels of actin. ( B ) The levels of PAD2 proteins in indicated C57BL/6 Th cells and PAD2-KO Th2 cells were examined with Western blotting using indicated antibodies. ( C ) At indicated time points during the differentiation of WT Th2 and Th17 cells, the level of PAD2 protein was examined with Western blotting. A parallel gel was probed with anti-tubulin for loading control. ( D and E ) WT Th cells were stimulated with anti-CD3, anti-CD28, and/or IL-2 in the absence ( D ) or presence ( E ) of cyclosporine A (CsA) or sotrastaurin (Sot) for 2 days. The level of PAD2 proteins was examined with Western blotting. Representative Western blots from 2 experiments are shown in B – D . ( F ) C57BL/6 effector Th cells were generated in the presence of AFM30a (left panel) or GSK199 (right panel) at indicated concentrations. The expression of indicated cytokines by the Th cells after restimulation with anti-CD3 is shown ( n = 3–4, 1-way ANOVA) . ( G – J ) Th cells obtained from WT DBA/1J ( G – J , n = 3–4), PAD2-KO ( G , I , and J , n = 3–5), or PAD4-KO ( H , n = 2) mice were differentiated into Th1, Th2, and Th17 cells for 5 days. The production of indicated cytokines after restimulation with anti-CD3 was measured with ELISA or real-time PCR ( G and H , 2-tailed Student’s t test for G ). Data from the same experiments are connected with lines in H . Whole cell extract from WT DBA/1J or PAD2-KO Th cells were examined with Western blotting using anti-citH3 ( I ). A parallel gel was probed with anti-H3 for loading controls. The normalized density of cit-H3 of indicated cells is shown in the dot blot of I . The transcript levels of PADs in restimulated WT DBA/1J, and PAD2-KO Th2 cells were quantified with real-time PCR and shown in J ( n = 3–4).

Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Generated, Expressing, Enzyme-linked Immunosorbent Assay, Dot Blot

Modulation of in vivo and in vitro differentiation of Th cells by global inhibition of citrullination. ( A ) Indicated C57BL/6 Th cells were left unstimulated (–) or restimulated with anti-CD3 (+) overnight. The level of cit-H3 was examined with Western blotting using anti–cit-H3. The normalized density of cit-H3 is shown in the bar graph ( n = 3, 1-way ANOVA). ( B and C ) C57BL/6 effector Th cells were generated in the presence of Cl-am at indicated concentrations for 5 days. Whole Th2 extract was examined with Western blotting using indicated antibodies. Representative blots and normalized density of cit-H3 from 2 experiments are shown in B . The expression of indicated cytokines by the Th cells after restimulation with anti-CD3 is shown in C ( n = 4, 1-way ANOVA) . ( D ) Primary human Th cells from 5 healthy donors were differentiated in vitro into Th2 or Th17 cells in the presence or absence of Cl-am (100 μM). The production of IL-4 and IL-17A after restimulation with anti-CD3 was quantified with ELISA. Data points from the same donors are connected with lines (1-tailed paired Student’s t test). ( E – I ) Allergic airway inflammation was induced in C57BL/6 mice ( n = 6 per group) in the absence or presence of Cl-am. Splenocytes were restimulated with ovalbumin for 72 hours. The levels of IL-4 and IL-17A in supernatant are shown in E . Imm, immunized; cha, challeneged. The levels of ovalbumin-specific (ova-specific) IgE and IgG1 in serum are shown in F . Representative H E staining of the lung tissue is shown in G . Scale bars: 100 μm. The total number of cells ( H ) and the percentage of eosinophils ( I ) in bronchial lavage are also shown. Statistical analysis for E , F , H , and I was performed with 2-tailed Student’s t test.
Figure Legend Snippet: Modulation of in vivo and in vitro differentiation of Th cells by global inhibition of citrullination. ( A ) Indicated C57BL/6 Th cells were left unstimulated (–) or restimulated with anti-CD3 (+) overnight. The level of cit-H3 was examined with Western blotting using anti–cit-H3. The normalized density of cit-H3 is shown in the bar graph ( n = 3, 1-way ANOVA). ( B and C ) C57BL/6 effector Th cells were generated in the presence of Cl-am at indicated concentrations for 5 days. Whole Th2 extract was examined with Western blotting using indicated antibodies. Representative blots and normalized density of cit-H3 from 2 experiments are shown in B . The expression of indicated cytokines by the Th cells after restimulation with anti-CD3 is shown in C ( n = 4, 1-way ANOVA) . ( D ) Primary human Th cells from 5 healthy donors were differentiated in vitro into Th2 or Th17 cells in the presence or absence of Cl-am (100 μM). The production of IL-4 and IL-17A after restimulation with anti-CD3 was quantified with ELISA. Data points from the same donors are connected with lines (1-tailed paired Student’s t test). ( E – I ) Allergic airway inflammation was induced in C57BL/6 mice ( n = 6 per group) in the absence or presence of Cl-am. Splenocytes were restimulated with ovalbumin for 72 hours. The levels of IL-4 and IL-17A in supernatant are shown in E . Imm, immunized; cha, challeneged. The levels of ovalbumin-specific (ova-specific) IgE and IgG1 in serum are shown in F . Representative H E staining of the lung tissue is shown in G . Scale bars: 100 μm. The total number of cells ( H ) and the percentage of eosinophils ( I ) in bronchial lavage are also shown. Statistical analysis for E , F , H , and I was performed with 2-tailed Student’s t test.

Techniques Used: In Vivo, In Vitro, Inhibition, Western Blot, Generated, Expressing, Enzyme-linked Immunosorbent Assay, Mouse Assay, Staining

30) Product Images from "Thrombocytopenia is associated with severe retinopathy of prematurity"

Article Title: Thrombocytopenia is associated with severe retinopathy of prematurity

Journal: JCI Insight

doi: 10.1172/jci.insight.99448

Platelet count in OIR and proof of concept. ( A ) Scheme of oxygen-induced retinopathy (OIR): C57BL/6 mouse pups are exposed to 75% oxygen from P7–P12 to induce retinal vessel loss and are returned to room air at P12–P17. Below the scheme, 2 representative retinal whole-mount images, including magnification area, from normoxia and OIR P17 pups stained with lectin (see magnification below). P17 OIR pups have pathologic neovascularization compared with normoxia pups. ( B ) Platelet counts in mouse pups at P7, P12, and P17 with (red) ( n = 10) or without (blue) ( n = 6) OIR. Platelet count was reduced at P17 OIR ( P = 0.0084). ( C ) Platelet counts were measured in whole blood after 0, 2, and 48 hours after anti-GPIbα antibody injection. A significant decrease in platelet count was seen at 2 and 48 hours after platelet depletion ( P = 0.0029 and P
Figure Legend Snippet: Platelet count in OIR and proof of concept. ( A ) Scheme of oxygen-induced retinopathy (OIR): C57BL/6 mouse pups are exposed to 75% oxygen from P7–P12 to induce retinal vessel loss and are returned to room air at P12–P17. Below the scheme, 2 representative retinal whole-mount images, including magnification area, from normoxia and OIR P17 pups stained with lectin (see magnification below). P17 OIR pups have pathologic neovascularization compared with normoxia pups. ( B ) Platelet counts in mouse pups at P7, P12, and P17 with (red) ( n = 10) or without (blue) ( n = 6) OIR. Platelet count was reduced at P17 OIR ( P = 0.0084). ( C ) Platelet counts were measured in whole blood after 0, 2, and 48 hours after anti-GPIbα antibody injection. A significant decrease in platelet count was seen at 2 and 48 hours after platelet depletion ( P = 0.0029 and P

Techniques Used: Staining, Injection

31) Product Images from "Comparison of RELMα and RELMβ Single- and Double-Gene-Deficient Mice Reveals that RELMα Expression Dictates Inflammation and Worm Expulsion in Hookworm Infection"

Article Title: Comparison of RELMα and RELMβ Single- and Double-Gene-Deficient Mice Reveals that RELMα Expression Dictates Inflammation and Worm Expulsion in Hookworm Infection

Journal: Infection and Immunity

doi: 10.1128/IAI.01479-15

Effect of Retnla or Retnlb genetic deletion on expression of RELMβ or RELMα, respectively. C57BL/6 (WT), Retnla −/− , and Retnlb −/− mice were injected with PBS or infected with N. brasiliensis for 3, 7, and 30 days. RELMα and RELMβ expression levels in the serum (A), BAL fluid (B), lung (C), and jejunum (D) were measured. Results are combined data from > 3 experiments ( n = 3 to 5 for naive mice, and n = 3 to 6 for mice infected at each time point). Statistical analysis was performed for each genotype compared to the WT at the same time point. *, P
Figure Legend Snippet: Effect of Retnla or Retnlb genetic deletion on expression of RELMβ or RELMα, respectively. C57BL/6 (WT), Retnla −/− , and Retnlb −/− mice were injected with PBS or infected with N. brasiliensis for 3, 7, and 30 days. RELMα and RELMβ expression levels in the serum (A), BAL fluid (B), lung (C), and jejunum (D) were measured. Results are combined data from > 3 experiments ( n = 3 to 5 for naive mice, and n = 3 to 6 for mice infected at each time point). Statistical analysis was performed for each genotype compared to the WT at the same time point. *, P

Techniques Used: Expressing, Mouse Assay, Injection, Infection

RELMα and RELMβ are expressed systemically and locally following N. brasiliensis infection. C57BL/6 mice were injected with PBS or infected with N. brasiliensis for 3, 7, and 30 days, followed by analysis of RELMα and RELMβ in the serum (A), bronchoalveolar lavage fluid (BALF) (B), lung (C), and jejunum (D) ( n = 4 to 5 per group). Experiments were repeated 3 to 5 times. Statistical analysis compared to naive mice was performed. *, P
Figure Legend Snippet: RELMα and RELMβ are expressed systemically and locally following N. brasiliensis infection. C57BL/6 mice were injected with PBS or infected with N. brasiliensis for 3, 7, and 30 days, followed by analysis of RELMα and RELMβ in the serum (A), bronchoalveolar lavage fluid (BALF) (B), lung (C), and jejunum (D) ( n = 4 to 5 per group). Experiments were repeated 3 to 5 times. Statistical analysis compared to naive mice was performed. *, P

Techniques Used: Infection, Mouse Assay, Injection

Function of RELMα and RELMβ in N. brasiliensis -induced inflammation and immune-mediated expulsion. (A and B) C57BL/6 (WT) and Retnla −/− Retnlb −/− mice were infected with N. brasiliensis and monitored daily for infection-induced weight loss ( n = 4 to 5 per group) (A) and mortality (for WT mice, n = 31 males and 9 females, and for Retnla −/− Retnlb −/− mice, n = 26 males and 8 females) (B). (C and D) BAL fluid cells were quantified for RBCs (C) and leukocytes (D) ( n = 4 to 8 per group). (E) Flow cytometric analysis of cell populations at day 9 postinfection ( n = 5 per group). (F and G) Parasites were recovered from the lung and small intestine at day 3 and the small intestine at days 6, 7, and 9 for worm quantification (F) and worm ATP measurements (G) ( n = 4 to 8 per group). (H) Fecal egg output over time ( n = 5 to 9 per group). (I and J) Real-time PCR analysis of mRNA from whole lung ( n = 3 to 5 per group) (I) and purified lung CD4 + T cells ( n = 6 to 8 per group) (J) from infected mice on day 7. *, P
Figure Legend Snippet: Function of RELMα and RELMβ in N. brasiliensis -induced inflammation and immune-mediated expulsion. (A and B) C57BL/6 (WT) and Retnla −/− Retnlb −/− mice were infected with N. brasiliensis and monitored daily for infection-induced weight loss ( n = 4 to 5 per group) (A) and mortality (for WT mice, n = 31 males and 9 females, and for Retnla −/− Retnlb −/− mice, n = 26 males and 8 females) (B). (C and D) BAL fluid cells were quantified for RBCs (C) and leukocytes (D) ( n = 4 to 8 per group). (E) Flow cytometric analysis of cell populations at day 9 postinfection ( n = 5 per group). (F and G) Parasites were recovered from the lung and small intestine at day 3 and the small intestine at days 6, 7, and 9 for worm quantification (F) and worm ATP measurements (G) ( n = 4 to 8 per group). (H) Fecal egg output over time ( n = 5 to 9 per group). (I and J) Real-time PCR analysis of mRNA from whole lung ( n = 3 to 5 per group) (I) and purified lung CD4 + T cells ( n = 6 to 8 per group) (J) from infected mice on day 7. *, P

Techniques Used: Mouse Assay, Infection, Flow Cytometry, Real-time Polymerase Chain Reaction, Purification

32) Product Images from "Expression of lymphotoxin-?? on antigen-specific T cells is required for DC function"

Article Title: Expression of lymphotoxin-?? on antigen-specific T cells is required for DC function

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20061968

Expression of LTβR ligands on OTII CD4 + T cells in response to OVA antigen. Purified OTII T cells were adoptively transferred into C57BL/6 mice, which were then immunized with OVA. Thy1.1 + OTII T cells in the draining LNs were analyzed for the expression of CD69, LTαβ, and LIGHT. The expression of LTαβ and LIGHT on WT OTII T cells is demonstrated with representative histograms from five mice (A), and a kinetic analysis of expression of CD69, LTαβ, and LIGHT was generated (B). The experiment was performed twice with similar results. Expression on respective knockout T cells was insignificant over background (not depicted).
Figure Legend Snippet: Expression of LTβR ligands on OTII CD4 + T cells in response to OVA antigen. Purified OTII T cells were adoptively transferred into C57BL/6 mice, which were then immunized with OVA. Thy1.1 + OTII T cells in the draining LNs were analyzed for the expression of CD69, LTαβ, and LIGHT. The expression of LTαβ and LIGHT on WT OTII T cells is demonstrated with representative histograms from five mice (A), and a kinetic analysis of expression of CD69, LTαβ, and LIGHT was generated (B). The experiment was performed twice with similar results. Expression on respective knockout T cells was insignificant over background (not depicted).

Techniques Used: Expressing, Purification, Mouse Assay, Generated, Knock-Out

LTαβ–LTβR interactions are required for optimal CD4 priming in vivo. (A) C57BL/6 mice received CFSE-labeled, OVA-specific WT- or LTβ −/− -OTII T cells, were immunized with OVA+LPS, or were left unimmunized. At 36 h after immunization, OTII CFSE content was assessed, comparing baseline CFSE content for each individual genotype. At day 5 after immunization, secretion of IFNγ from OTII T cells was assessed by intracellular FACS. (B) C57BL/6 mice received OVA-specific WT-OTII T cells, were treated with huIgG or LTβR-Ig, and were immunized with OVA+LPS. At day 7, the frequency of IFNγ + OTII T cells was assessed. The experiments were performed twice with three to five mice per group in each experiment.
Figure Legend Snippet: LTαβ–LTβR interactions are required for optimal CD4 priming in vivo. (A) C57BL/6 mice received CFSE-labeled, OVA-specific WT- or LTβ −/− -OTII T cells, were immunized with OVA+LPS, or were left unimmunized. At 36 h after immunization, OTII CFSE content was assessed, comparing baseline CFSE content for each individual genotype. At day 5 after immunization, secretion of IFNγ from OTII T cells was assessed by intracellular FACS. (B) C57BL/6 mice received OVA-specific WT-OTII T cells, were treated with huIgG or LTβR-Ig, and were immunized with OVA+LPS. At day 7, the frequency of IFNγ + OTII T cells was assessed. The experiments were performed twice with three to five mice per group in each experiment.

Techniques Used: In Vivo, Mouse Assay, Labeling, FACS

Function of DCs conditioned by adoptively transferred WT-, LTβ −/− -, or LIGHT −/− -OTII T cells. (A) C57BL/6 mice received WT-, LTβ −/− -, or LIGHT −/− -OTII T cells and were immunized, and LN cell suspensions were gated on CD4 and Thy1.1 and analyzed for CD69 and CD25 at 36 h after immunization, with filled histograms representing Thy1.1 T cells transferred into WT-unimmunized mice. (B) C57BL/6 mice received OVA-specific WT- (filled squares, filled circles), LTβ −/− - (empty circles), or LIGHT −/− -OTII T cells (empty triangles), or were immunized or left unimmunized (filled circles). At 36 h after immunization, draining LN DCs were plated with OTII responder T cells and incubated at 37°C for 72 h. (C) Proliferation results from B are represented as a SI. CPM derived from OTII T cells cocultured with DCs was divided by CPM derived from OTII T cells cocultured with the same number of DC-depleted cells at the same cell input number for each individual group (internally controlled). Groups are responder OTII T cells stimulated by DCs conditioned in vivo by WT- (black bars), LTβ −/− - (gray bars), or LIGHT −/− -OTII T cells (open bars) and compared with DCs from unimmunized mice that received WT-OTII T cells (speckled bars). (D) A similar experiment was performed using DCs conditioned by WT-OTII versus LTβ −/− -OTII to stimulate naive responder OTI T cells using 15,000 or 30,000 DCs per well (black and gray bars, respectively). (E) IFNγ secretion by OTI CD8 + T cells from was evaluated by ELISA using 15,000 or 30,000 DCs per well (black and gray bars, respectively). OTII responder experiments were performed four times using DCs pooled from seven individual animals. OTI responder experiments were performed two times using DCs pooled from seven individual animals.
Figure Legend Snippet: Function of DCs conditioned by adoptively transferred WT-, LTβ −/− -, or LIGHT −/− -OTII T cells. (A) C57BL/6 mice received WT-, LTβ −/− -, or LIGHT −/− -OTII T cells and were immunized, and LN cell suspensions were gated on CD4 and Thy1.1 and analyzed for CD69 and CD25 at 36 h after immunization, with filled histograms representing Thy1.1 T cells transferred into WT-unimmunized mice. (B) C57BL/6 mice received OVA-specific WT- (filled squares, filled circles), LTβ −/− - (empty circles), or LIGHT −/− -OTII T cells (empty triangles), or were immunized or left unimmunized (filled circles). At 36 h after immunization, draining LN DCs were plated with OTII responder T cells and incubated at 37°C for 72 h. (C) Proliferation results from B are represented as a SI. CPM derived from OTII T cells cocultured with DCs was divided by CPM derived from OTII T cells cocultured with the same number of DC-depleted cells at the same cell input number for each individual group (internally controlled). Groups are responder OTII T cells stimulated by DCs conditioned in vivo by WT- (black bars), LTβ −/− - (gray bars), or LIGHT −/− -OTII T cells (open bars) and compared with DCs from unimmunized mice that received WT-OTII T cells (speckled bars). (D) A similar experiment was performed using DCs conditioned by WT-OTII versus LTβ −/− -OTII to stimulate naive responder OTI T cells using 15,000 or 30,000 DCs per well (black and gray bars, respectively). (E) IFNγ secretion by OTI CD8 + T cells from was evaluated by ELISA using 15,000 or 30,000 DCs per well (black and gray bars, respectively). OTII responder experiments were performed four times using DCs pooled from seven individual animals. OTI responder experiments were performed two times using DCs pooled from seven individual animals.

Techniques Used: Mouse Assay, Incubation, Derivative Assay, In Vivo, Enzyme-linked Immunosorbent Assay

Expression of CD40L on OTII T cells is required for DC function, and agonistic anti-CD40 or -LTβR treatment can rescue DCs conditioned by LTβ −/− -OTII or CD40L −/− -OTII T cells, respectively. (A) C57BL/6 mice received CFSE-labeled WT- or CD40L −/− -OTII T cells, were immunized with OVA-LPS s.c., or were left unimmunized. CFSE content and the expression of CD69 and CD25 were measured by FACS at 36 h after immunization. (B) Draining LN DCs from mice immunized as in A were plated with OTII responder T cells and incubated at 37°C for 72 h. Filled circles, WT-OTII T cells; empty circles, CD40L −/− -OTII T cells. (C) C57BL/6 mice received LTβ −/− - (i and ii) or CD40L −/− -OTII T cells (iii and iv), were immunized with OVA-LPS s.c., and were treated with the indicated agonistic Ab or control Ab. Purified draining LN DCs (20,000 DCs for i and ii; 10,000 DCs for iii and iv) were plated with OTI and OTII responder T cells and incubated at 37°C for 72 h. The experiments were performed two times with seven mice per group, except the experiment in C (i–ii), which was performed three times with seven mice per group.
Figure Legend Snippet: Expression of CD40L on OTII T cells is required for DC function, and agonistic anti-CD40 or -LTβR treatment can rescue DCs conditioned by LTβ −/− -OTII or CD40L −/− -OTII T cells, respectively. (A) C57BL/6 mice received CFSE-labeled WT- or CD40L −/− -OTII T cells, were immunized with OVA-LPS s.c., or were left unimmunized. CFSE content and the expression of CD69 and CD25 were measured by FACS at 36 h after immunization. (B) Draining LN DCs from mice immunized as in A were plated with OTII responder T cells and incubated at 37°C for 72 h. Filled circles, WT-OTII T cells; empty circles, CD40L −/− -OTII T cells. (C) C57BL/6 mice received LTβ −/− - (i and ii) or CD40L −/− -OTII T cells (iii and iv), were immunized with OVA-LPS s.c., and were treated with the indicated agonistic Ab or control Ab. Purified draining LN DCs (20,000 DCs for i and ii; 10,000 DCs for iii and iv) were plated with OTI and OTII responder T cells and incubated at 37°C for 72 h. The experiments were performed two times with seven mice per group, except the experiment in C (i–ii), which was performed three times with seven mice per group.

Techniques Used: Expressing, Mouse Assay, Labeling, FACS, Incubation, Purification

33) Product Images from "Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel ?IIb-specific ?IIb?3 antagonist"

Article Title: Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel ?IIb-specific ?IIb?3 antagonist

Journal: Blood

doi: 10.1182/blood-2008-08-169243

RUC-1 inhibits aggregation of human platelets and hαIIb/mβ3 murine platelets, but not WT mouse or rat platelets . PRP was isolated from whole blood of WT Sprague-Dawley rats, WT C57Bl/6 mice, healthy human volunteers, or hybrid hαIIb/mβ3
Figure Legend Snippet: RUC-1 inhibits aggregation of human platelets and hαIIb/mβ3 murine platelets, but not WT mouse or rat platelets . PRP was isolated from whole blood of WT Sprague-Dawley rats, WT C57Bl/6 mice, healthy human volunteers, or hybrid hαIIb/mβ3

Techniques Used: Isolation, Mouse Assay

34) Product Images from "Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases"

Article Title: Targeting IL-17A/glucocorticoid synergy to CSF3 expression in neutrophilic airway diseases

Journal: JCI Insight

doi: 10.1172/jci.insight.132836

C3G reduces emphysema and restores DEX sensitivity in elastase-induced neutrophilic COPD. ( A ) Eight-week-old WT C57BL/6 female mice ( n = 5 mice per group) were subjected to the elastase-induced COPD model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). One week after the last challenge, total cell and neutrophil counts in the BAL were quantified ( B ), and representative BAL samples were prepared by cytospin and lung tissues were stained with H E ( C ). All scale bars (red): 100 μm. ( D ) Mean linear intercept (MLI) of airway space was measured by ImageJ. ( E ) The relative abundances of the indicated mRNAs isolated from lung tissue were determined by real-time PCR. ( F ) The total collagen content in lung tissue was quantified. COPD, chronic obstructive pulmonary disease; AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. For MLI ( n = 20), Kruskal-Wallis test (nonparametric) was performed, followed by Dunn’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.
Figure Legend Snippet: C3G reduces emphysema and restores DEX sensitivity in elastase-induced neutrophilic COPD. ( A ) Eight-week-old WT C57BL/6 female mice ( n = 5 mice per group) were subjected to the elastase-induced COPD model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). One week after the last challenge, total cell and neutrophil counts in the BAL were quantified ( B ), and representative BAL samples were prepared by cytospin and lung tissues were stained with H E ( C ). All scale bars (red): 100 μm. ( D ) Mean linear intercept (MLI) of airway space was measured by ImageJ. ( E ) The relative abundances of the indicated mRNAs isolated from lung tissue were determined by real-time PCR. ( F ) The total collagen content in lung tissue was quantified. COPD, chronic obstructive pulmonary disease; AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. For MLI ( n = 20), Kruskal-Wallis test (nonparametric) was performed, followed by Dunn’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.

Techniques Used: Mouse Assay, Staining, Isolation, Real-time Polymerase Chain Reaction

Disruption of IL-17A/DEX synergistic effect on CSF3 expression renders DEX sensitivity in HDM-CFA type-17 acute asthma model. ( A ) Eight-week-old WT C57BL/6 female mice ( n = 5 per group) were subjected to the HDM-CFA acute severe asthma model. Isotype antibody (Control), DEX, anti-CSF3, and anti–IL-17A neutralizing antibodies or combined DEX and antibodies were administered to the mice (as described in Methods). Twenty-four hours after challenging, total cell and neutrophil counts in the BAL were quantified. ( B ) mRNA expression of lung tissues was quantified by real-time PCR. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.
Figure Legend Snippet: Disruption of IL-17A/DEX synergistic effect on CSF3 expression renders DEX sensitivity in HDM-CFA type-17 acute asthma model. ( A ) Eight-week-old WT C57BL/6 female mice ( n = 5 per group) were subjected to the HDM-CFA acute severe asthma model. Isotype antibody (Control), DEX, anti-CSF3, and anti–IL-17A neutralizing antibodies or combined DEX and antibodies were administered to the mice (as described in Methods). Twenty-four hours after challenging, total cell and neutrophil counts in the BAL were quantified. ( B ) mRNA expression of lung tissues was quantified by real-time PCR. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.

Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

Small-molecule IL-17A blocker C3G alleviates neutrophilia and improves DEX sensitivity in HDM-CFA type-17 acute asthma model. ( A ) Chemical structure of C3G. ( B ) Human and mouse ASMCs (hASMCs and mASMCs) were treated for 24 hours with IL-17A in the presence of DMSO (vehicle control) or the indicated concentrations of C3G. The concentrations of CXCL1 (GROα in humans and KC in mice) in the culture medium were measured by ELISA. Results are presented as a percentage of the amount of CXCL1 in the culture medium of DMSO-treated cells, which was set at 100%. Data are representative of 3 independent experiments. Error bars represent the SEM of technical replicates. ( C and D ) Eight-week-old WT C57BL/6 female mice ( n = 5 per group) were subjected to the HDM-CFA acute asthma model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). Twenty-four hours after challenging, total and neutrophil counts in the BAL were quantified ( C ). Representative BAL cells were prepared by cytospin and lung tissues were stained with H E ( D ). All scale bars (red): 100 μm. ( E ) mRNA expression of lung tissues was quantified by real-time PCR. ( F and G ) OVA 323–339 -specific Th17 cells were adoptively transferred into 8-week-old female C57BL/6 mice ( n = 5 mice per treatment group), which were treated as described in Methods. Twenty-four hours after the last challenge, total cell and neutrophil counts in the BAL were quantified. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.
Figure Legend Snippet: Small-molecule IL-17A blocker C3G alleviates neutrophilia and improves DEX sensitivity in HDM-CFA type-17 acute asthma model. ( A ) Chemical structure of C3G. ( B ) Human and mouse ASMCs (hASMCs and mASMCs) were treated for 24 hours with IL-17A in the presence of DMSO (vehicle control) or the indicated concentrations of C3G. The concentrations of CXCL1 (GROα in humans and KC in mice) in the culture medium were measured by ELISA. Results are presented as a percentage of the amount of CXCL1 in the culture medium of DMSO-treated cells, which was set at 100%. Data are representative of 3 independent experiments. Error bars represent the SEM of technical replicates. ( C and D ) Eight-week-old WT C57BL/6 female mice ( n = 5 per group) were subjected to the HDM-CFA acute asthma model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). Twenty-four hours after challenging, total and neutrophil counts in the BAL were quantified ( C ). Representative BAL cells were prepared by cytospin and lung tissues were stained with H E ( D ). All scale bars (red): 100 μm. ( E ) mRNA expression of lung tissues was quantified by real-time PCR. ( F and G ) OVA 323–339 -specific Th17 cells were adoptively transferred into 8-week-old female C57BL/6 mice ( n = 5 mice per treatment group), which were treated as described in Methods. Twenty-four hours after the last challenge, total cell and neutrophil counts in the BAL were quantified. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay, Staining, Expressing, Real-time Polymerase Chain Reaction

C3G ameliorates airway remodeling and mitigates DEX resistance in the HDM-CFA type-17 chronic asthma model. ( A ) Eight-week-old WT C57BL/6 female mice ( n = 5 mice per group) were subjected to the HDM-CFA chronic asthma model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). Twenty-four hours after the last challenge, total cell and neutrophil counts in the BAL were quantified ( B ), and representative BAL cells were prepared by cytospin and lung tissues were subjected to histochemical staining as indicated ( C ). All scale bars (red): 100 μm. ( D and E ) PAS score (PAS-positive cells/total epithelial cells) and total collagen content of lung tissue were measured. ( F ) mRNA expression of lung tissues was quantified by real-time PCR. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.
Figure Legend Snippet: C3G ameliorates airway remodeling and mitigates DEX resistance in the HDM-CFA type-17 chronic asthma model. ( A ) Eight-week-old WT C57BL/6 female mice ( n = 5 mice per group) were subjected to the HDM-CFA chronic asthma model. PBS (Control), DEX, C3G, or a combination of DEX and C3G were administered to the mice (as described in Methods). Twenty-four hours after the last challenge, total cell and neutrophil counts in the BAL were quantified ( B ), and representative BAL cells were prepared by cytospin and lung tissues were subjected to histochemical staining as indicated ( C ). All scale bars (red): 100 μm. ( D and E ) PAS score (PAS-positive cells/total epithelial cells) and total collagen content of lung tissue were measured. ( F ) mRNA expression of lung tissues was quantified by real-time PCR. AU, fold induction relative to unchallenged control mice. Data represent mean ± SEM. One-way ANOVA was performed, followed by Tukey’s multiple-comparisons test. The multiplicity-adjusted P values were calculated for the indicated comparisons. The above data are representative of 3 independent experiments.

Techniques Used: Mouse Assay, Staining, Expressing, Real-time Polymerase Chain Reaction

35) Product Images from "A?42 neurotoxicity in primary co-cultures: Effect of apoE isoform and A? conformation"

Article Title: A?42 neurotoxicity in primary co-cultures: Effect of apoE isoform and A? conformation

Journal:

doi: 10.1016/j.neurobiolaging.2006.05.024

Oligomeric Aβ42, but not fibrillar Aβ42, induced a dose-dependent increase in neurotoxicity in the presence of apoE2-TR glia (A), apoE3-TR glia (B) and apoE4-TR glia (C). Cortical neurons from WT C57Bl/6 mice were co-cultured with glia
Figure Legend Snippet: Oligomeric Aβ42, but not fibrillar Aβ42, induced a dose-dependent increase in neurotoxicity in the presence of apoE2-TR glia (A), apoE3-TR glia (B) and apoE4-TR glia (C). Cortical neurons from WT C57Bl/6 mice were co-cultured with glia

Techniques Used: Mouse Assay, Cell Culture

Oligomeric Aβ42, but not fibrillar Aβ42, induced a dose- and time-dependent increase in neurotoxicity in the presence of WT glia (A) and KO glia (B). Cortical neurons from WT C57BL/6 mice were co-cultured with glial (~95% astrocytes) cells
Figure Legend Snippet: Oligomeric Aβ42, but not fibrillar Aβ42, induced a dose- and time-dependent increase in neurotoxicity in the presence of WT glia (A) and KO glia (B). Cortical neurons from WT C57BL/6 mice were co-cultured with glial (~95% astrocytes) cells

Techniques Used: Mouse Assay, Cell Culture

Neurons co-cultured with apoE4-expressing glia showed the highest oligomeric Aβ42-induced neurotoxicity. Cortical neurons from WT C57BL/6 mice were co-cultured with glia (~95% astrocytes) from WT (■), apoE-KO ( ), apoE2-TR ( ), apoE3-TR
Figure Legend Snippet: Neurons co-cultured with apoE4-expressing glia showed the highest oligomeric Aβ42-induced neurotoxicity. Cortical neurons from WT C57BL/6 mice were co-cultured with glia (~95% astrocytes) from WT (■), apoE-KO ( ), apoE2-TR ( ), apoE3-TR

Techniques Used: Cell Culture, Expressing, Mouse Assay

36) Product Images from "Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity"

Article Title: Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity

Journal: Cell

doi: 10.1016/j.cell.2020.09.058

Induction of Trained Immunity Inhibits Tumor Growth (A) Experimental scheme. (B) C57BL/6 WT mice received a single i.p. injection of β-glucan or PBS and 7 days thereafter, mice were subcutaneously inoculated with B16-F10 melanoma cells. Shown on the left, tumor volume was monitored for another 14 days after tumor inoculation. Shown on the right, tumor weight at the end of the experiment (n = 6 mice in the PBS group; n = 7 mice in the β-glucan group). (C) C57BL/6 WT mice received a single i.p. injection of β-glucan or PBS and 7 days thereafter, mice were inoculated with LLC cells. Tumor volume is shown (n = 7 mice in the PBS group; n = 5 mice in the β-glucan group). (D) C57BL/6 WT mice received β-glucan or PBS and 7 days thereafter, mice were inoculated with B16-F10 melanoma cells. Flow-cytometric analysis for immune cells that are infiltrated in the B16-F10 melanoma tumors was performed at the end of the experiment. Frequencies of myeloid cells (CD45 + CD11b + ), neutrophils (CD45 + CD11b + Ly6g + Ly6c − ), monocytes (CD45 + CD11b + Ly6g − Ly6c + ), and macrophages (CD45 + CD11b + F4/80 + ) within leukocytes (CD45 + ) are shown (n = 6 mice per group). (E) Rag1 −/− mice received a single i.p. injection of β-glucan or PBS and 7 days thereafter, mice were inoculated with B16-F10 melanoma cells. Shown on the left is tumor volume; on the right is tumor weight at the end of the experiment (n = 8 mice in the PBS group; n = 5 mice in the β-glucan group). (F) Rag1 −/− mice received β-glucan or PBS and 7 days thereafter, mice were inoculated with LLC cells. Tumor volume is shown (n = 16 mice in the PBS group; n = 12 mice in the β-glucan group). Data are presented as mean ± SEM; n.s., non-significant; ∗ p
Figure Legend Snippet: Induction of Trained Immunity Inhibits Tumor Growth (A) Experimental scheme. (B) C57BL/6 WT mice received a single i.p. injection of β-glucan or PBS and 7 days thereafter, mice were subcutaneously inoculated with B16-F10 melanoma cells. Shown on the left, tumor volume was monitored for another 14 days after tumor inoculation. Shown on the right, tumor weight at the end of the experiment (n = 6 mice in the PBS group; n = 7 mice in the β-glucan group). (C) C57BL/6 WT mice received a single i.p. injection of β-glucan or PBS and 7 days thereafter, mice were inoculated with LLC cells. Tumor volume is shown (n = 7 mice in the PBS group; n = 5 mice in the β-glucan group). (D) C57BL/6 WT mice received β-glucan or PBS and 7 days thereafter, mice were inoculated with B16-F10 melanoma cells. Flow-cytometric analysis for immune cells that are infiltrated in the B16-F10 melanoma tumors was performed at the end of the experiment. Frequencies of myeloid cells (CD45 + CD11b + ), neutrophils (CD45 + CD11b + Ly6g + Ly6c − ), monocytes (CD45 + CD11b + Ly6g − Ly6c + ), and macrophages (CD45 + CD11b + F4/80 + ) within leukocytes (CD45 + ) are shown (n = 6 mice per group). (E) Rag1 −/− mice received a single i.p. injection of β-glucan or PBS and 7 days thereafter, mice were inoculated with B16-F10 melanoma cells. Shown on the left is tumor volume; on the right is tumor weight at the end of the experiment (n = 8 mice in the PBS group; n = 5 mice in the β-glucan group). (F) Rag1 −/− mice received β-glucan or PBS and 7 days thereafter, mice were inoculated with LLC cells. Tumor volume is shown (n = 16 mice in the PBS group; n = 12 mice in the β-glucan group). Data are presented as mean ± SEM; n.s., non-significant; ∗ p

Techniques Used: Mouse Assay, Injection

37) Product Images from "Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina"

Article Title: Photoreceptor cells are major contributors to diabetes-induced oxidative stress and local inflammation in the retina

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1314575110

Genetic degeneration of photoreceptors prevents diabetes-induced generation of superoxide in mouse retina. Photoreceptor degeneration was induced by genetic deficiency of opsin ( opsin −/− ) in C57BL/6 mice. ( A ) Compared with wild-type control
Figure Legend Snippet: Genetic degeneration of photoreceptors prevents diabetes-induced generation of superoxide in mouse retina. Photoreceptor degeneration was induced by genetic deficiency of opsin ( opsin −/− ) in C57BL/6 mice. ( A ) Compared with wild-type control

Techniques Used: Mouse Assay

38) Product Images from "Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct"

Article Title: Cell-specific qRT-PCR of renal epithelial cells reveals a novel innate immune signature in murine collecting duct

Journal: American Journal of Physiology - Renal Physiology

doi: 10.1152/ajprenal.00512.2016

Defb1 RNA in situ hybridization on wild-type C57BL/6 kidney section showing expression of Defb1 (purple, arrows; A ) and VATPase-E1 antibody (green, arrowhead; B ). C : overlay. *Cells positive for both VATPase (green) and Defb1 (purple). Magnification: ×60.
Figure Legend Snippet: Defb1 RNA in situ hybridization on wild-type C57BL/6 kidney section showing expression of Defb1 (purple, arrows; A ) and VATPase-E1 antibody (green, arrowhead; B ). C : overlay. *Cells positive for both VATPase (green) and Defb1 (purple). Magnification: ×60.

Techniques Used: RNA In Situ Hybridization, Expressing

39) Product Images from "Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4"

Article Title: Indoleamine 2,3-dioxygenase is a critical resistance mechanism in antitumor T cell immunotherapy targeting CTLA-4

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20130066

IDO impairs antitumor effects of anti-CTLA4 in the context of B16 tumors engineered to overexpress IDO. (A) Expression of GFP (black line) or IDO/GFP (gray) in B16F10 cells determined by flow cytometry. WT B16F10 cells were used for comparison (dotted line). (B) In vitro growth rate of GFP-B16F10 and IDO/GFP-B16F10 cells. (C) Mean growth of GFP-B16F10 and IDO/GFP-B16F10 tumors in naive and irradiated (450 cGy) mice. (D) Tumor-free survival curves for C57BL/6 mice challenged with GFP-B16F10 or IDO/GFP-B16F10 tumor cells i.d. and treated with anti–CTLA-4 or anti-CLTA-4/1MT. Data represent cumulative results from two independent experiments with five mice/group. Statistical significance was determined with Log-Rank test (*, P
Figure Legend Snippet: IDO impairs antitumor effects of anti-CTLA4 in the context of B16 tumors engineered to overexpress IDO. (A) Expression of GFP (black line) or IDO/GFP (gray) in B16F10 cells determined by flow cytometry. WT B16F10 cells were used for comparison (dotted line). (B) In vitro growth rate of GFP-B16F10 and IDO/GFP-B16F10 cells. (C) Mean growth of GFP-B16F10 and IDO/GFP-B16F10 tumors in naive and irradiated (450 cGy) mice. (D) Tumor-free survival curves for C57BL/6 mice challenged with GFP-B16F10 or IDO/GFP-B16F10 tumor cells i.d. and treated with anti–CTLA-4 or anti-CLTA-4/1MT. Data represent cumulative results from two independent experiments with five mice/group. Statistical significance was determined with Log-Rank test (*, P

Techniques Used: Expressing, Flow Cytometry, Cytometry, In Vitro, Irradiation, Mouse Assay

Anti–CTLA-4/1MT treatment increases the ratio of effector T cells to T reg cells in tumor and elicits a tumor-specific T cell response. B16F10 tumors from untreated, anti–CTLA-4–treated, and anti–CTLA-4/1MT–treated C57BL/6 mice were harvested 15 d after tumor challenge and analyzed by flow cytometry for their content of effector T cells and T reg cells. (A) Tumor weights. (B) Percentage of CD8 + , CD4 + Foxp3 − , and CD4 + Foxp3 + T cells of total CD45 + cells. (C) Immune infiltrate analysis expressed as a percentage of total CD45 + cells. (D) Percentage of CD11b + Gr-1 + MDSCs of total CD45 + cells and representative dot plots. (E) Ratio of CD4 + Foxp3 − to CD4 + Foxp3 + cells and CD8 + to CD4 + Foxp3 + cells. (F) Absolute numbers of CD8 + T cells per gram of tumor. (G) Frequency of CD8 + GrB + T cells of total CD45 + cells. TILs were restimulated for 4 h with PMA/Ionomycin (H) or overnight with DCs loaded with B16F10 tumor lysate or DCs with MC38 lysate as a nonmelanoma control tumor (I), and production of IFN-γ was determined by flow cytometry. Data were analyzed by two-way ANOVA (A, B, E, and F; *, P
Figure Legend Snippet: Anti–CTLA-4/1MT treatment increases the ratio of effector T cells to T reg cells in tumor and elicits a tumor-specific T cell response. B16F10 tumors from untreated, anti–CTLA-4–treated, and anti–CTLA-4/1MT–treated C57BL/6 mice were harvested 15 d after tumor challenge and analyzed by flow cytometry for their content of effector T cells and T reg cells. (A) Tumor weights. (B) Percentage of CD8 + , CD4 + Foxp3 − , and CD4 + Foxp3 + T cells of total CD45 + cells. (C) Immune infiltrate analysis expressed as a percentage of total CD45 + cells. (D) Percentage of CD11b + Gr-1 + MDSCs of total CD45 + cells and representative dot plots. (E) Ratio of CD4 + Foxp3 − to CD4 + Foxp3 + cells and CD8 + to CD4 + Foxp3 + cells. (F) Absolute numbers of CD8 + T cells per gram of tumor. (G) Frequency of CD8 + GrB + T cells of total CD45 + cells. TILs were restimulated for 4 h with PMA/Ionomycin (H) or overnight with DCs loaded with B16F10 tumor lysate or DCs with MC38 lysate as a nonmelanoma control tumor (I), and production of IFN-γ was determined by flow cytometry. Data were analyzed by two-way ANOVA (A, B, E, and F; *, P

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

Anti–CTLA-4 and 1MT synergize to mediate tumor rejection. Treatment schedule (A), Kaplan-Meier survival curves (B), mean tumor size (C) and individual tumor growths (D) for C57BL/6 mice challenged with B16F10 melanoma cells i.d. and treated with anti–CTLA-4 and/or 1MT. The numbers of mice/group rejecting tumors was: untreated (0/10 mice), anti–CTLA-4 (2/10), anti–CTLA-4/L-1MT (6/10), and anti–CTLA-4/D-1MT (5/10). Kaplan-Meier survival curves for C57BL/6 mice challenged with B16BL6 and treated with anti–CTLA-4, Gvax, and/or 1MT. 1MT was administered in the drinking water (E) or as time-release subcutaneous pellets (F). Data shown are pooled from two (C and D) or three (B, E, and F) independent experiments with five mice/group. Statistical significance was evaluated by Log-Rank Test(B, E, and F; *, P
Figure Legend Snippet: Anti–CTLA-4 and 1MT synergize to mediate tumor rejection. Treatment schedule (A), Kaplan-Meier survival curves (B), mean tumor size (C) and individual tumor growths (D) for C57BL/6 mice challenged with B16F10 melanoma cells i.d. and treated with anti–CTLA-4 and/or 1MT. The numbers of mice/group rejecting tumors was: untreated (0/10 mice), anti–CTLA-4 (2/10), anti–CTLA-4/L-1MT (6/10), and anti–CTLA-4/D-1MT (5/10). Kaplan-Meier survival curves for C57BL/6 mice challenged with B16BL6 and treated with anti–CTLA-4, Gvax, and/or 1MT. 1MT was administered in the drinking water (E) or as time-release subcutaneous pellets (F). Data shown are pooled from two (C and D) or three (B, E, and F) independent experiments with five mice/group. Statistical significance was evaluated by Log-Rank Test(B, E, and F; *, P

Techniques Used: Mouse Assay

Anti–CTLA-4/1MT antitumor effect is T cell dependent. (A) Mean tumor growth and tumor-free survival curves for C57BL/6 mice challenged with B16F10 tumors i.d. and treated with anti–CTLA-4/1MT plus depleting antibodies for IFN-γ, CD8, CD4, or NK/NKT, or a corresponding dose of IgG isotype control (IgG). P-values are for two-way ANOVA and Log-Rank test (*, P
Figure Legend Snippet: Anti–CTLA-4/1MT antitumor effect is T cell dependent. (A) Mean tumor growth and tumor-free survival curves for C57BL/6 mice challenged with B16F10 tumors i.d. and treated with anti–CTLA-4/1MT plus depleting antibodies for IFN-γ, CD8, CD4, or NK/NKT, or a corresponding dose of IgG isotype control (IgG). P-values are for two-way ANOVA and Log-Rank test (*, P

Techniques Used: Mouse Assay

40) Product Images from "EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization"

Article Title: EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20132038

Expression of SAP family adaptors in various NK cell populations. (A) NKPs, iNK cells, and mNK cells were sorted from bone marrow of C57BL/6 mice by first gating on forward scatter channel (FSC)- and side scatter channel (SSC)-low cells and then on lineage (Lin) − CD122 + cells. NKPs are NK1.1 − CD49b − , whereas iNK and mNK cells are NK1.1 + , CD49b − , and NK1.1 + CD49b + , respectively. (B) RNAs from purified NK cell populations in bone marrow, spleen mNK, and splenic NK cells from poly I:C–primed mice (poly I:C), LAK cells, and splenic B cells were subjected to reverse transcription and real-time PCR with gene-specific primers for Sh2d1a (SAP), Sh2d1b1 (EAT-2), Sh2d1b2 (ERT), or Gapdh . Cross threshold (CT) values were normalized to Gapdh for each cell type and are relative to values for LAK cells. The resulting values (ΔCT) are shown. Mean values with error bars and standard deviations of duplicates from a representative experiment are shown. Shown is a representative of 4 independent experiments. (C) Normalized RNA expression for Sh2d1a (SAP), Sh2d1b1 (EAT-2), and Sh2d1b2 (ERT) in total, Ly49C/I + , Ly49C/I − , Ly49H + , or Ly49H − resting splenic NK cells, or in NK cells from mice infected for 1 (D1) or 7 (D7) days with mouse cytomegalovirus (MCMV), were obtained from the Immgen consortium. Values for splenic follicular B cells (B fo) are shown as control. Details on data generation are available at www.immgen.org .
Figure Legend Snippet: Expression of SAP family adaptors in various NK cell populations. (A) NKPs, iNK cells, and mNK cells were sorted from bone marrow of C57BL/6 mice by first gating on forward scatter channel (FSC)- and side scatter channel (SSC)-low cells and then on lineage (Lin) − CD122 + cells. NKPs are NK1.1 − CD49b − , whereas iNK and mNK cells are NK1.1 + , CD49b − , and NK1.1 + CD49b + , respectively. (B) RNAs from purified NK cell populations in bone marrow, spleen mNK, and splenic NK cells from poly I:C–primed mice (poly I:C), LAK cells, and splenic B cells were subjected to reverse transcription and real-time PCR with gene-specific primers for Sh2d1a (SAP), Sh2d1b1 (EAT-2), Sh2d1b2 (ERT), or Gapdh . Cross threshold (CT) values were normalized to Gapdh for each cell type and are relative to values for LAK cells. The resulting values (ΔCT) are shown. Mean values with error bars and standard deviations of duplicates from a representative experiment are shown. Shown is a representative of 4 independent experiments. (C) Normalized RNA expression for Sh2d1a (SAP), Sh2d1b1 (EAT-2), and Sh2d1b2 (ERT) in total, Ly49C/I + , Ly49C/I − , Ly49H + , or Ly49H − resting splenic NK cells, or in NK cells from mice infected for 1 (D1) or 7 (D7) days with mouse cytomegalovirus (MCMV), were obtained from the Immgen consortium. Values for splenic follicular B cells (B fo) are shown as control. Details on data generation are available at www.immgen.org .

Techniques Used: Expressing, Mouse Assay, Purification, Real-time Polymerase Chain Reaction, RNA Expression, Infection

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Isolation:

Article Title: A Group A Streptococcus ADP-Ribosyltransferase Toxin Stimulates a Protective Interleukin 1β-Dependent Macrophage Immune Response
Article Snippet: .. BMDMs were isolated from the femurs and tibia of 8-to-12-week-old WT C57BL/6 mice (Jackson Laboratory) and casp-1/11 −/− (provided by R. Flavell), Asc−/− , or Nlrp3−/− (provided by J. Bertin of Millennium Pharmaceuticals) and IL-1β−/− (provided by D. Chaplin) C57BL/6 mice. .. Femurs and tibia were flushed with room-temperature Dulbecco’s modified Eagle’s medium (DMEM), and precursor cells were cultured in DMEM–20% fetal bovine serum (FBS), 20% L929 cell-conditioned media, 1% nonessential amino acids, 1% sodium pyruvate, and 1% penicillin-streptomycin and grown at 37°C in 5% CO2 for 7 days, with changes of media on day 4.

Infection:

Article Title: Zika virus infection damages the testes in mice
Article Snippet: .. Mouse infection experiments WT C57BL/6 mice were purchased commercially (Jackson Laboratories) and congenic Rag1 -/- mice were bred at Washington University in a pathogen-free facility. .. Congenic Axl -/- mice were described previously .

Mouse Assay:

Article Title: Tumor cell–intrinsic EPHA2 suppresses antitumor immunity by regulating PTGS2 (COX-2)
Article Snippet: .. All WT C57BL/6 mice were purchased from The Jackson Laboratory and/or bred at the University of Pennsylvania. .. Kras-LSL-G12D/+ ; Trp53-LSL-R172H/+ , Pdx1-Cre, and Rosa-LSL-YFP (KPCY) mice ( , ) were bred in-house, backcrossed for over 10 generations with C57BL/6J mice (Jackson Laboratories), and assessed at the DartMouse Speed Congenic Core facility at the Geisel School of Medicine at Dartmouth College (Hanover, New Hampshire, USA).

Article Title: TF protein of Sindbis virus antagonizes host type I interferon responses in a palmitoylation-dependent manner
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Article Snippet: .. For PTC sort, WT C57BL/6 mice (stock no.000664; Jackson Laboratory) were used. .. Kidney single cell suspension was prepared from Aqp2-Cre+ tdT+/− mice to enrich PCs, V-ATPase-Cre+ tdT+/− mice to enrich ICs, and WT C57BL/6 mice to enrich PTCs.

Article Title: Zika virus infection damages the testes in mice
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Article Snippet: .. BMDMs were isolated from the femurs and tibia of 8-to-12-week-old WT C57BL/6 mice (Jackson Laboratory) and casp-1/11 −/− (provided by R. Flavell), Asc−/− , or Nlrp3−/− (provided by J. Bertin of Millennium Pharmaceuticals) and IL-1β−/− (provided by D. Chaplin) C57BL/6 mice. .. Femurs and tibia were flushed with room-temperature Dulbecco’s modified Eagle’s medium (DMEM), and precursor cells were cultured in DMEM–20% fetal bovine serum (FBS), 20% L929 cell-conditioned media, 1% nonessential amino acids, 1% sodium pyruvate, and 1% penicillin-streptomycin and grown at 37°C in 5% CO2 for 7 days, with changes of media on day 4.

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    The Jackson Laboratory c57bl 6 wt control mice
    Impaired IgG and GC responses in CD154 TG CD22 −/− mice. ( A ) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22 −/− , CD154 TG , and CD154 TG CD22 −/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. ( B ) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT <t>C57BL/6</t> and 6 mo-old MLR lpr mice were used as negative and positive controls, respectively. ( C ) Impaired IgG responses to a TD Ag. WT ( n = 3), CD22 −/− ( n = 4), CD154 TG ( n = 6), and CD154 TG CD22 −/− ( n = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220 + B cells (red) and GC GL7 + B220 + B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22 −/− mice, and detectable GL7 + B220 + B cells within the follicles of CD154 TG mice, but not in CD154 TG CD22 −/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p
    C57bl 6 Wt Control Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Jackson Laboratory strain matched wt c57bl 6 mice
    LTB 4 is required for IL-1β production. A) Quantification of IL-1β and B) Il1b mRNA expression in the skin of <t>C57BL/6,</t> Ltb4r1 −/− , C57BL/6 treated with a topical ointment containing LTB 4 or C57BL/6 treated with a topical ointment containing BLT1 antagonist U-75302 24h after MRSA infection. C) IVIS scanning of pIL1DsRed mice treated with a topical ointment containing BLT1 antagonist U-75032 or vehicle. TNF-α quantification in D) peritoneal macrophages treated with LTB 4 or BLT1 antagonist before MRSA infection and E) skin biopsy from wild-type (WT) or Ltb4r1 −/− mice after 24h MRSA infection. F) IL-1β quantification from human neutrophils isolated from healthy blood donors treated with LTB 4 and LPS. Data are the mean ± SEM of 3-10 mice. *p
    Strain Matched Wt C57bl 6 Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The Jackson Laboratory wt c57bl 6 male mice
    Protective efficacy of anti-JEV MAbs in mice. (A and B) Four- to 5-week-old male <t>C57BL/6</t> mice were passively administered 10 µg of the indicated (A) mouse or (B) human MAb via intraperitoneal injection 1 day prior to inoculation with 10 2 FFU of JEV-Nakayama via the subcutaneous route. JEV-31 ( n = 9), JEV-106 ( n = 8), JEV-143 ( n = 8), and JEV-169 ( n = 10) provided complete protection against lethality. JEV-27 ( n = 8), JEV-128 ( n = 9), and JEV-131 ( n = 9) provided partial protection compared to the isotype control MAbs. (C and D) Three-week-old male C57BL/6 mice were passively administered 10 µg of the indicated MAb as described above 1 day prior to inoculation with 10 3 FFU of (C) JEV-MAR 859 (JEV-31, n = 8; JEV-131, n = 9; JEV-169, n = 8) or (D) JEV-2372/79 (JEV-31, n = 9; JEV-106, n = 9; JEV-131, n = 9; JEV-169, n = 9). (E and F) Two hundred fifty micrograms of the indicated MAb was administered 5 days postinfection to (E) 4- to 5-week-old mice infected with 10 2 FFU of JEV-Nakayama (JEV-31, n = 9; JEV-106, n = 9; JEV-143, n = 9; JEV-169, n = 9; hJEV-75, n = 8) or (F) 3-week-old mice infected with 10 3 FFU of JEV-2372/79 (JEV-31, n = 10; JEV-131, n = 9; JEV-143, n = 9; JEV-169, n = 10; hJEV-75, n = 9). Data are pooled from at least two independent experiments. Survival was analyzed for each MAb compared to the isotype control MAb by the log rank test. *, P
    Wt C57bl 6 Male Mice, supplied by The Jackson Laboratory, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    IgA and IgG2b levels are elevated in TCRδ −/− mice. Levels of Ig isotypes were determined by ELISA in serum (A), fecal pellet (B), and peritoneal lavage (C) from WT (C57BL/6) and TCRδ −/− mice. Data are expressed as means±standard error of the mean (n=3 mice). n.s., not significant. * p

    Journal: Immune Network

    Article Title: Murine γδ T Cells Render B Cells Refractory to Commitment of IgA Isotype Switching

    doi: 10.4110/in.2018.18.e25

    Figure Lengend Snippet: IgA and IgG2b levels are elevated in TCRδ −/− mice. Levels of Ig isotypes were determined by ELISA in serum (A), fecal pellet (B), and peritoneal lavage (C) from WT (C57BL/6) and TCRδ −/− mice. Data are expressed as means±standard error of the mean (n=3 mice). n.s., not significant. * p

    Article Snippet: Animals and genotyping of TCRδ knockout mice Wild-type (WT) C57BL/6 (Daehan Biolink, Seoul, Korea) and TCRδ−/− mice ( ) (JAX stock #002119, Jackson Lab) were maintained on an 8:16-h light:dark cycle in an animal environmental control chamber (Daehan Biolink).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Impaired IgG and GC responses in CD154 TG CD22 −/− mice. ( A ) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22 −/− , CD154 TG , and CD154 TG CD22 −/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. ( B ) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT C57BL/6 and 6 mo-old MLR lpr mice were used as negative and positive controls, respectively. ( C ) Impaired IgG responses to a TD Ag. WT ( n = 3), CD22 −/− ( n = 4), CD154 TG ( n = 6), and CD154 TG CD22 −/− ( n = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220 + B cells (red) and GC GL7 + B220 + B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22 −/− mice, and detectable GL7 + B220 + B cells within the follicles of CD154 TG mice, but not in CD154 TG CD22 −/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p

    Journal: PLoS ONE

    Article Title: Amplified B Lymphocyte CD40 Signaling Drives Regulatory B10 Cell Expansion in Mice

    doi: 10.1371/journal.pone.0022464

    Figure Lengend Snippet: Impaired IgG and GC responses in CD154 TG CD22 −/− mice. ( A ) Serum IgM and IgG levels of 4 and 12 mo-old WT, CD22 −/− , CD154 TG , and CD154 TG CD22 −/− mice. Symbols represent serum concentrations for individual mice as determined by ELISA, with means indicated by horizontal bars. ( B ) Serum autoAbs reactive with dsDNA, ssDNA, or histone proteins in 12 mo-old mice. ELISA OD values for IgM (upper panels) and IgG (lower panels) autoAbs are shown for individual mice, with means indicated by horizontal bars. Sera from 2 mo-old WT C57BL/6 and 6 mo-old MLR lpr mice were used as negative and positive controls, respectively. ( C ) Impaired IgG responses to a TD Ag. WT ( n = 3), CD22 −/− ( n = 4), CD154 TG ( n = 6), and CD154 TG CD22 −/− ( n = 8) mice were immunized with DNP-KLH in adjuvant on day 0, and boosted on day 21. The graph shows mean (±SEM) DNP-specific IgG levels as determined by ELISA. Images on the right represent immunofluorescence staining of frozen spleen sections from all genotypes harvested 7 days after the boost phase of DNP-KLH immunization. Merged images show the presence of B220 + B cells (red) and GC GL7 + B220 + B cells (yellow). Enlarged regions from these sections indicate typical GC structures present within the follicles of WT and CD22 −/− mice, and detectable GL7 + B220 + B cells within the follicles of CD154 TG mice, but not in CD154 TG CD22 −/− mice (representative regions are shown for comparison). (A–C) Means significantly different from WT are indicated by asterisks (*p≤0.05, **p≤0.01), and between other indicated groups by crosses (†p

    Article Snippet: C57BL/6 WT control mice were purchased from either The Jackson Laboratory (Bar Harbor, ME) or NCI Frederick (Bethesda, MD).

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

    LTB 4 is required for IL-1β production. A) Quantification of IL-1β and B) Il1b mRNA expression in the skin of C57BL/6, Ltb4r1 −/− , C57BL/6 treated with a topical ointment containing LTB 4 or C57BL/6 treated with a topical ointment containing BLT1 antagonist U-75302 24h after MRSA infection. C) IVIS scanning of pIL1DsRed mice treated with a topical ointment containing BLT1 antagonist U-75032 or vehicle. TNF-α quantification in D) peritoneal macrophages treated with LTB 4 or BLT1 antagonist before MRSA infection and E) skin biopsy from wild-type (WT) or Ltb4r1 −/− mice after 24h MRSA infection. F) IL-1β quantification from human neutrophils isolated from healthy blood donors treated with LTB 4 and LPS. Data are the mean ± SEM of 3-10 mice. *p

    Journal: bioRxiv

    Article Title: Leukotriene B4 licenses inflammasome activation to enhance skin host defense

    doi: 10.1101/2020.02.03.932129

    Figure Lengend Snippet: LTB 4 is required for IL-1β production. A) Quantification of IL-1β and B) Il1b mRNA expression in the skin of C57BL/6, Ltb4r1 −/− , C57BL/6 treated with a topical ointment containing LTB 4 or C57BL/6 treated with a topical ointment containing BLT1 antagonist U-75302 24h after MRSA infection. C) IVIS scanning of pIL1DsRed mice treated with a topical ointment containing BLT1 antagonist U-75032 or vehicle. TNF-α quantification in D) peritoneal macrophages treated with LTB 4 or BLT1 antagonist before MRSA infection and E) skin biopsy from wild-type (WT) or Ltb4r1 −/− mice after 24h MRSA infection. F) IL-1β quantification from human neutrophils isolated from healthy blood donors treated with LTB 4 and LPS. Data are the mean ± SEM of 3-10 mice. *p

    Article Snippet: Eighteen-week-old female or male BLT1−/− (B6.129S4-Ltb4r1tm1Adl /J , LysMcre, MMDTR, and strain-matched WT C57BL/6 mice were purchased from Jackson Labs (Bar Harbor, ME USA).

    Techniques: Expressing, Infection, Mouse Assay, Isolation

    Protective efficacy of anti-JEV MAbs in mice. (A and B) Four- to 5-week-old male C57BL/6 mice were passively administered 10 µg of the indicated (A) mouse or (B) human MAb via intraperitoneal injection 1 day prior to inoculation with 10 2 FFU of JEV-Nakayama via the subcutaneous route. JEV-31 ( n = 9), JEV-106 ( n = 8), JEV-143 ( n = 8), and JEV-169 ( n = 10) provided complete protection against lethality. JEV-27 ( n = 8), JEV-128 ( n = 9), and JEV-131 ( n = 9) provided partial protection compared to the isotype control MAbs. (C and D) Three-week-old male C57BL/6 mice were passively administered 10 µg of the indicated MAb as described above 1 day prior to inoculation with 10 3 FFU of (C) JEV-MAR 859 (JEV-31, n = 8; JEV-131, n = 9; JEV-169, n = 8) or (D) JEV-2372/79 (JEV-31, n = 9; JEV-106, n = 9; JEV-131, n = 9; JEV-169, n = 9). (E and F) Two hundred fifty micrograms of the indicated MAb was administered 5 days postinfection to (E) 4- to 5-week-old mice infected with 10 2 FFU of JEV-Nakayama (JEV-31, n = 9; JEV-106, n = 9; JEV-143, n = 9; JEV-169, n = 9; hJEV-75, n = 8) or (F) 3-week-old mice infected with 10 3 FFU of JEV-2372/79 (JEV-31, n = 10; JEV-131, n = 9; JEV-143, n = 9; JEV-169, n = 10; hJEV-75, n = 9). Data are pooled from at least two independent experiments. Survival was analyzed for each MAb compared to the isotype control MAb by the log rank test. *, P

    Journal: mBio

    Article Title: Mouse and Human Monoclonal Antibodies Protect against Infection by Multiple Genotypes of Japanese Encephalitis Virus

    doi: 10.1128/mBio.00008-18

    Figure Lengend Snippet: Protective efficacy of anti-JEV MAbs in mice. (A and B) Four- to 5-week-old male C57BL/6 mice were passively administered 10 µg of the indicated (A) mouse or (B) human MAb via intraperitoneal injection 1 day prior to inoculation with 10 2 FFU of JEV-Nakayama via the subcutaneous route. JEV-31 ( n = 9), JEV-106 ( n = 8), JEV-143 ( n = 8), and JEV-169 ( n = 10) provided complete protection against lethality. JEV-27 ( n = 8), JEV-128 ( n = 9), and JEV-131 ( n = 9) provided partial protection compared to the isotype control MAbs. (C and D) Three-week-old male C57BL/6 mice were passively administered 10 µg of the indicated MAb as described above 1 day prior to inoculation with 10 3 FFU of (C) JEV-MAR 859 (JEV-31, n = 8; JEV-131, n = 9; JEV-169, n = 8) or (D) JEV-2372/79 (JEV-31, n = 9; JEV-106, n = 9; JEV-131, n = 9; JEV-169, n = 9). (E and F) Two hundred fifty micrograms of the indicated MAb was administered 5 days postinfection to (E) 4- to 5-week-old mice infected with 10 2 FFU of JEV-Nakayama (JEV-31, n = 9; JEV-106, n = 9; JEV-143, n = 9; JEV-169, n = 9; hJEV-75, n = 8) or (F) 3-week-old mice infected with 10 3 FFU of JEV-2372/79 (JEV-31, n = 10; JEV-131, n = 9; JEV-143, n = 9; JEV-169, n = 10; hJEV-75, n = 9). Data are pooled from at least two independent experiments. Survival was analyzed for each MAb compared to the isotype control MAb by the log rank test. *, P

    Article Snippet: WT C57BL/6 male mice (4 to 5 weeks old; Jackson Laboratories) were inoculated with 102 FFU of JEV-Nakayama subcutaneously in the footpad.

    Techniques: Mouse Assay, Injection, Infection