Journal: Neural Regeneration Research

Article Title: Spliceosomal GTPase Eftud2 regulates microglial activation and polarization

doi: 10.4103/1673-5374.347739

Figure Lengend Snippet: Primers used for real-time PCR

Article Snippet: The following antibodies were used overnight at 4°C: rabbit anti-Eftud2 (1:200, Abcam, Cambridge, UK, Cat# ab72456, RRID: AB_1268731), mouse anti-Ki67 (1:400, Becton Dickinson and Company, Franklin Lake, NJ, USA, Cat# 550609, RRID: AB_393778), goat anti-Iba-1 (1:400, Abcam, Cat# ab5076, RRID: AB_2224402), rabbit anti-Arg1 (1:400, Proteintech, Chicago, IL, USA, Cat# 16001-1-AP, RRID:AB_2289842), rabbit anti-iNOS (1:200, Cell Signaling Technology, Boston, MA, USA, Cat# 13120, RRID: AB_2687529), mouse anti-GFAP (1:400, Sigma-Aldrich, Cat# G3893, RRID: AB_477010), and mouse anti-NeuN (1:400, Abcam, Cat# ab104224, RRID: AB_10711040).

Techniques: Sequencing

Journal: Neural Regeneration Research

Article Title: Spliceosomal GTPase Eftud2 regulates microglial activation and polarization

doi: 10.4103/1673-5374.347739

Figure Lengend Snippet: Eftud2 deficiency promotes activation of anti-inflammatory phenotype microglia. (A) Iba-1 (green, Alexa Fluor 488), Ki-67 (red, Alexa Fluor568), and iNOS (blue, Alexa Fluor 405) triple immunostaining in cortex of Eftud2 f/f and cKO mice after 4-OH-TAM injection. Iba-1 + iNOS + and Iba-1 + iNOS + Ki67 + cells in Eftud2 cKO mice were not significantly different from controls. Scale bars: 20 μm. (B) Iba-1 and iNOS double-positive microglial cell count. (C) Iba-1, Ki-67 and iNOS triple-positive microglial cell count. (D) Iba-1 (green, Alexa Fluor 488), Ki-67 (red, Alexa Fluor568), and iNOS (blue, Alexa Fluor 405) triple immunostaining in cortex of Eftud2 f/f and cKO mice after TAM injection. Iba-1 + Arg1 + and Iba-1 + Arg1 + Ki67 + cells in Eftud2 cKO mice were significantly increased compared with those in controls. Scale bars: 20 μm. (E) Iba-1 and Arg1 double-positive microglial cell count. (F) Iba-1, Ki-67, and Arg1 triple-positive microglial cell count. Data are presented as mean ± SEM. At least three mice were used for each genotype. **** P < 0.0001 (two-tailed unpaired Student’s t -test). 4-OH-TAM: 4-OH-Tamoxifen; Arg1: arginase-1; cKO: conditional knockout; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; iNOS: inducible nitric oxide synthase; ns: not significant.

Article Snippet: The following antibodies were used overnight at 4°C: rabbit anti-Eftud2 (1:200, Abcam, Cambridge, UK, Cat# ab72456, RRID: AB_1268731), mouse anti-Ki67 (1:400, Becton Dickinson and Company, Franklin Lake, NJ, USA, Cat# 550609, RRID: AB_393778), goat anti-Iba-1 (1:400, Abcam, Cat# ab5076, RRID: AB_2224402), rabbit anti-Arg1 (1:400, Proteintech, Chicago, IL, USA, Cat# 16001-1-AP, RRID:AB_2289842), rabbit anti-iNOS (1:200, Cell Signaling Technology, Boston, MA, USA, Cat# 13120, RRID: AB_2687529), mouse anti-GFAP (1:400, Sigma-Aldrich, Cat# G3893, RRID: AB_477010), and mouse anti-NeuN (1:400, Abcam, Cat# ab104224, RRID: AB_10711040).

Techniques: Activation Assay, Triple Immunostaining, Injection, Cell Counting, Two Tailed Test, Knock-Out, Binding Assay

Journal: Neural Regeneration Research

Article Title: Spliceosomal GTPase Eftud2 regulates microglial activation and polarization

doi: 10.4103/1673-5374.347739

Figure Lengend Snippet: Eftud2 knockdown promotes anti-inflammatory phenotype activation in BV2 cells (A) RT-PCR analysis of Eftud2 expression in BV2 cells infected with siRNA against Eftud2 (SiEftud2). (B–E) RT-PCR analysis of iNOS, TNF-α, Arg1, and IL-10 mRNA levels normalized to negative control (NC) after infection with SiEftud2. (F) Fluorescent microspheres (red) and DAPI (blue) in BV2 cells after infection with SiEftud2. Eftud2-knockout microglia engulfed more beads than controls. Scale bars: 5 μm. (G, H) Quantification of microglial phagocytosis. Data are presented as mean ± SEM. At least three mice were used for each genotype. * P < 0.05, *** P < 0.001, **** P < 0.0001 (two-tailed unpaired Student’s t -test). Arg1: Arginase-1; DAPI: 4',6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; IL-10: interleukin-10; iNOS: inducible nitric oxide synthase; NC: negative control; RT-PCR: real-time polymerase chain reaction; siEftud2: short interfering RNA against Eftud2; TNF-α: tumor necrosis factor-α; ns: not significant.

Article Snippet: The following antibodies were used overnight at 4°C: rabbit anti-Eftud2 (1:200, Abcam, Cambridge, UK, Cat# ab72456, RRID: AB_1268731), mouse anti-Ki67 (1:400, Becton Dickinson and Company, Franklin Lake, NJ, USA, Cat# 550609, RRID: AB_393778), goat anti-Iba-1 (1:400, Abcam, Cat# ab5076, RRID: AB_2224402), rabbit anti-Arg1 (1:400, Proteintech, Chicago, IL, USA, Cat# 16001-1-AP, RRID:AB_2289842), rabbit anti-iNOS (1:200, Cell Signaling Technology, Boston, MA, USA, Cat# 13120, RRID: AB_2687529), mouse anti-GFAP (1:400, Sigma-Aldrich, Cat# G3893, RRID: AB_477010), and mouse anti-NeuN (1:400, Abcam, Cat# ab104224, RRID: AB_10711040).

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Negative Control, Knock-Out, Two Tailed Test, Binding Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Spliceosomal GTPase Eftud2 regulates microglial activation and polarization

doi: 10.4103/1673-5374.347739

Figure Lengend Snippet: Eftud2 regulates proinflammatory and anti-inflammatory phenotype activation of microglia involved in inflammatory responses via the NF-κB pathway. (A–D) RT-PCR analysis of iNOS, TNFα, Arg1, and IL-10 mRNA levels in NC and SiEftud2 microglial cells after exposure to LPS for the indicated times. (E) Fluorescent microspheres (red) and DAPI (blue) in NC and SiEftud2 microglial cells after exposure to LPS. Eftud2-knockout microglia engulfed more beads than NC groups. Scale bar: 5 μm. (F, G) Quantification of microglial phagocytosis. (H) Immunoblots of TLR4, MyD88, p65, p-p65 and β-actin proteins in NC, and SiEftud2. (I) Quantification of TLR4, MyD88, p65, and p-p65 levels normalized to β-actin. (J) RT-PCR analysis of TLR4, MyD88, and p65 mRNA levels in NC and SiEftud2 microglial cells after exposure to LPS. Data are presented as mean ± SEM. At least three independent experiments were repeated. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two-tailed unpaired Student’s t -test). Arg1: Arginase-1; DAPI: 4',6-diamidino-2-phenylindole; Eftud2: elongation factor Tu GTP binding domain protein 2; Iba-1: ionized calcium-binding adaptor molecule 1; IL-10: interleukin-10; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; MyD88: myeloid differentiation primary response gene 88; NC: negative control; NF-κB: nuclear transcription factor-κB; ns: not significant; RT-PCR: real-time polymerase chain reaction; siEftud2: short interfering RNA against Eftud2; TLR4: Toll-like receptor 4; TNF-α: tumor necrosis factor-α.

Article Snippet: The following antibodies were used overnight at 4°C: rabbit anti-Eftud2 (1:200, Abcam, Cambridge, UK, Cat# ab72456, RRID: AB_1268731), mouse anti-Ki67 (1:400, Becton Dickinson and Company, Franklin Lake, NJ, USA, Cat# 550609, RRID: AB_393778), goat anti-Iba-1 (1:400, Abcam, Cat# ab5076, RRID: AB_2224402), rabbit anti-Arg1 (1:400, Proteintech, Chicago, IL, USA, Cat# 16001-1-AP, RRID:AB_2289842), rabbit anti-iNOS (1:200, Cell Signaling Technology, Boston, MA, USA, Cat# 13120, RRID: AB_2687529), mouse anti-GFAP (1:400, Sigma-Aldrich, Cat# G3893, RRID: AB_477010), and mouse anti-NeuN (1:400, Abcam, Cat# ab104224, RRID: AB_10711040).

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Knock-Out, Western Blot, Two Tailed Test, Binding Assay, Negative Control, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Scientific Reports

Article Title: In vitro and in silico studies of 7′′,8′′-buddlenol D anti-inflammatory lignans from Carallia brachiata as p38 MAP kinase inhibitors

doi: 10.1038/s41598-023-30475-5

Figure Lengend Snippet: The effects of 1 and 2 on inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) protein expression. ( A ) Representative immunoblots showing LPS-induced RAW264.7 macrophages treated with 1 and 2 . The cropped blots were used in the figure. The protein lysates of each treatment group from the same experiment were performed simultaneously on the same gel for each protein detection. Full-length uncropped blots were shown in supplementary information data (Figure ). Densitometric histograms showing ( B ) iNOS and ( C ) COX2 in LPS-induced RAW264.7 macrophages. Dexamethasone was used as a positive control. Data were expressed as relative intensity when compared with LPS-induced groups and were represented as the mean ± standard error of the mean from three independent experiments (n = 3). * p < 0.05 vs. the LPS-induced group.

Article Snippet: The blots were blocked in 5% bovine serum albumin and probed with primary antibodies: anti-iNOS (ab15323), anti-COX2 (CST#2282), anti-phospho-ERK (CST#4376), anti-ERK (CST#4695), anti-phospho-JNK (CST#4668), anti-JNK (CST#9252), anti-phospho-p38 (CST#4511), anti-p38 (CST#9212), and followed by incubation with a horse radish peroxidase-conjugated secondary antibody.

Techniques: Expressing, Western Blot, Positive Control

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: NO production and NOS expression in CD8 + T cells. A, CD8 + T cells were activated with anti-CD3/CD28 Dynabeads (or SIINFEKL peptide, when using OT-I CD8 + T cells) for 1 to 4 days in 21%, 5%, and 1% O 2 . After activation, NO production and NOS expression were analyzed. B, NO production determined by extracellular quantification of nitrites (NO 2 − , a NO byproduct) in mouse CD8 + T cells activated for 3 days ( N = 11–13). C, Western blot analysis of NOS using a panNOS antibody (all isoforms detected) in lysates of mouse CD8 + T cell activated for 3 days. Results normalized to total protein stain (TPS, top) and representative blot (bottom; N = 3). D, Time course qRT-PCR analysis of Nos2 and Nos3 mRNA expression in activated mouse CD8 + T cells ( N = 3–9). E, Western blot analysis of NOS2 protein levels in mouse OT-I CD8 + T cells treated or untreated with 50 μmol/L FG-4592 and activated for 3 days in 1% O 2 with increasing amounts of SIINFEKL peptide (0.001, 0.1, 1, and 1000 ng/mL). Quantification normalized to TPS (top) and representative blot (bottom). BMDMs polarized to M1 with 100 U/mL LPS were used as positive control for NOS2 expression ( N = 3). F, NO production as determined by extracellular quantification of nitrites in human T cells cultured for 1 day in 21% and 1% O 2 with or without anti-CD3/CD28 beads ( N = 4). G, Time course qRT-PCR analysis of NOS2 and NOS3 mRNA levels in activated human CD8 + T cells. NOS1 mRNA levels were under the detection limit ( N = 5–8). H, Western blot analysis of panNOS (antibody detecting all NOS isoforms) and NOS3 in human CD8+ T cells. HUVEC cells were used as positive control and PPIB was used as loading control (representative of N = 3). Apart from panel D and G , each data point represents an independent animal and results are shown as median ± interquartile range (IQR). *, P < 0.05; Wilcoxon matched-pairs signed-rank test.

Article Snippet: Mouse protein extracts (25–40 μg) were probed with primary antibodies against panNOS (CST #2977), NOS2 (CST #13120), and hypoxia-inducible factor (HIF)-1α (Novus NB-100–449) and detected using infrared-labeled donkey anti-rabbit (926–32213, LI-COR) and goat anti-mouse (926–68070, LI-COR) secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Expressing, Activation Assay, Western Blot, Staining, Quantitative RT-PCR, Positive Control, Cell Culture

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: In vitro characterization of Nos2 KO CD8 + T cells. A, After activation in 1% O 2 with anti-CD3/CD28 beads, NOS2 expression was determined by Western blot in Nos2 fl/fl (WT, gray) and Nos2 fl/fl dLck Cre (NOS2 KO , orange) cells. Representative blot (top) and quantification normalized to total protein stain (bottom; N = 2). B, WT and NOS2 KO CD8 + T cells were activated for 3 days in 21% or 1% O 2 , and NO production was determined by the extracellular nitrite concentration ( N = 5–8). C, WT and NOS2 KO mouse CD8 + T cells were activated for 72 hours in 21%, 5%, or 1% O 2 . Viable CD8 + T-cell number was determined by flow cytometry using count beads (left); cell proliferation assessed with CTV staining and expressed as division index (right; N = 8–18). D, Proportion of CD62L − CD44 + in cells activated as in C (left) and representative FACS plots for 1% O 2 activated cells (right; N = 11–18). E, Heat map illustrating expression of markers of differentiation determined by flow cytometry in CD8 + T cells activated for 72 hours in 21%, 5%, or 1% O 2 . Increased and reduced expression of proteins are shown in gray and orange, respectively. Rows represent averaged z-scores ( N = 11–18). F, Seahorse metabolic analysis of mouse T cells activated for 3 days in 1% O 2 , as determined by OCR and ECAR after injection of anti-CD3/CD28 beads or antibodies, oligomycin (O), FCCP (F), or rotenone+antimycin A (R+A; left). Effect of T-cell activation on T-cell OCR and ECAR was determined by % change from baseline following injection of anti-CD3/CD28 beads or antibodies. Seahorse analysis was conducted in a hypoxia chamber set to 3%O 2 ( N = 8). G, OT-I CD8 + T cells activated for 3 days in 1% O 2 were cocultured with 10,000 OVA-expressing B16-F10 tumor cells at different effector:target (E:T) ratios. Cytotoxicity was assessed with Alamar blue assay after 14 to 18 hours of coculture at 21% O 2 . A nonlinear regression [(agonist) vs. normalized response] was used to determine dose–response curves (shaded areas: 95% confidence intervals; N = 4–6). H, CD8 + T cells were activated for 6 days and incubated for 48 hours in 1% O 2 before being loaded with calcein-AM and cocultured with mouse endothelial cells in a transwell system. mCCL19 and mCCL21 were added to the lower chamber as chemoattractant. Calcein signal corresponding to T cells migrating through the endothelial barrier was assessed after 3 hours of coculture in a plate reader ( N = 7–9). All results [median ± interquartile range (IQR)] are pooled from a minimum of two independent experiments, and each data point from panels B , F (right), and G and H represent an independent animal. ns, P > 0.05; *, P < 0.05; **, P < 0.01; Mann–Whitney test relative to respective WT control.

Article Snippet: Mouse protein extracts (25–40 μg) were probed with primary antibodies against panNOS (CST #2977), NOS2 (CST #13120), and hypoxia-inducible factor (HIF)-1α (Novus NB-100–449) and detected using infrared-labeled donkey anti-rabbit (926–32213, LI-COR) and goat anti-mouse (926–68070, LI-COR) secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: In Vitro, Activation Assay, Expressing, Western Blot, Staining, Concentration Assay, Flow Cytometry, Injection, Alamar Blue Assay, Incubation, MANN-WHITNEY

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: Tumor growth in animals lacking Nos2 expression in T cells. A, Tumor growth model. 5×10 5 MC38 or B16-F10-OVA were subcutaneously injected in Nos2 fl/fl (WT) Nos2 fl/fl dlck Cre (NOS2 KO ) animals. On day 10 after tumor inoculation, peripheral blood and tumors were processed to single-cell suspensions and analyzed by flow cytometry. Tumor growth was monitored until day 30. B, MC38 (top) and B16-F10-OVA (bottom) tumor growth data. Tumor growth curves in WT and NOS2 KO animals; thin lines represent individual animals and thick line represents an exponential (Malthusian) growth curve (left). Survival curves using 500 mm 3 as threshold (right). Results pooled from two independent experiments ( N = 18–26 animals per group). C, Immune composition was analyzed by flow cytometry on peripheral blood of animals bearing MC38 (top; N = 6–8) and B16-F10-OVA (bottom; N = 13–18) tumors for 10 days. Results expressed as cells per milliliter of blood; median ± interquartile range (IQR). D, Representative flow cytometry plots from CD4 + and CD8 + T-cell infiltration in B16-F10-OVA tumors collected on day 10 following inoculation in WT and NOS2 KO animals (top). Immune cell infiltration in B16-F10-OVA analyzed by flow cytometry and expressed as counts per million CD45 + cells (bottom). Cells pregated on live, singlet, CD45 + events ( N = 9, median ± IQR). Each data point represents an individual animal. ns, P > 0.05; *, P < 0.05; **, P < 0.05; log-rank (Mantel–Cox) test relative to WT animals ( B ) and Mann–Whitney test relative to WT control ( C and D ).

Article Snippet: Mouse protein extracts (25–40 μg) were probed with primary antibodies against panNOS (CST #2977), NOS2 (CST #13120), and hypoxia-inducible factor (HIF)-1α (Novus NB-100–449) and detected using infrared-labeled donkey anti-rabbit (926–32213, LI-COR) and goat anti-mouse (926–68070, LI-COR) secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Expressing, Injection, Flow Cytometry, MANN-WHITNEY

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: Antitumor function and tissue infiltration capacity of NOS2 KO OT-I T cells. A, ACT model. C57BL/6j mice were injected subcutaneously with 1×10 6 OVA-expressing B16-F10 tumor cells, and 4 days later were lymphodepleted with 300 mg/kg CPA. Mice bearing tumors for 7 days were then intraperitoneally injected with 1×10 6 of 4 days activated WT or NOS2 KO OT-I cells. Tumor growth was monitored every 2 to 3 days until day 60. B, B16-F10-OVA tumor growth after ACT. Tumor growth curves after No ACT or ACT with VC or NOS2 KO OT-I cells; vertical dotted lines represent day of ACT, thin lines represent individual animals, and thick lines represent an exponential (Malthusian) growth curve (left). Survival curves using 500 mm 3 as threshold (right; N = 9–20 animals per group). C, Tumor infiltration model. C57BL/6j mice were injected subcutaneously with 1×10 6 OVA-expressing B16-F10 tumor cells, and 11 days later were lymphodepleted with CPA. Mice bearing tumors for 14 days were then intraperitoneally injected with Nos2 fl/fl (WT) and Nos2 fl/fl dlck Cre (NOS2 KO ) OT-I CD8 + T cells (1×10 6 each in 1:1 NOS2 KO :WT ratio). Spleen, peripheral blood, liver, and tumor were harvested on day 19 and processed to single-cell suspensions for flow cytometric analysis. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. D, Total OT-I T-cell expansion in all analyzed tissues expressed as a ratio between NOS2 KO and WT cell counts (gray horizontal line represents the NOS2 KO /WT ratio at the time of injection; N = 22, median ± interquartile range [IQR]). E , Percentage of cells expressing granzyme B (GZMB) within CD8 + T cells in all tissues analyzed by flow cytometry on day 19 (bottom; N = 19–22, median ± IQR). F, Representative FACS plots (left) and flow cytometry analysis (right) of percentage of CD44 + CD8 + T cells in peripheral blood on day 19 ( N = 19–22, median ± IQR). Results are pooled from at least two independent experiments, and each data point represents an independent animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001; log-rank (Mantel–Cox) test relative to WT animals ( B ), one sample t test relative to 1 ( D ) and Wilcoxon matched-pairs signed-rank test relative to WT control ( E and F ).

Article Snippet: Mouse protein extracts (25–40 μg) were probed with primary antibodies against panNOS (CST #2977), NOS2 (CST #13120), and hypoxia-inducible factor (HIF)-1α (Novus NB-100–449) and detected using infrared-labeled donkey anti-rabbit (926–32213, LI-COR) and goat anti-mouse (926–68070, LI-COR) secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: Injection, Expressing, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: Infiltration of Tumors Is Regulated by T cell–Intrinsic Nitric Oxide Synthesis

doi: 10.1158/2326-6066.CIR-22-0387

Figure Lengend Snippet: In vivo activation and recall response of NOS2 KO OT-I CD8 + T cells. A, Scheme of in vivo T-cell activation and recall response model. C57BL/6j mice were injected intraperitoneally with 1×10 6 naïve Nos2 fl/fl (WT) and Nos2 fl/fl dlck Cre (NOS2 KO ) OT-I CD8 + T (1:1 NOS2 KO :WT ratio). The next day, mouse BMDMs differentiated for 7 days and polarized with LPS for 24 hours were pulsed with SIINFEKL peptide for 4 hours prior to intraperitoneal injection. Peripheral blood was sampled at days 7 and 10 after BMDM transfer and analyzed by flow cytometry. SIINFEKL-pulsed BMDMs (or PBS controls) were administered again on day 30. At day 37, the spleen, inguinal lymph nodes, and a liver portion were harvested and analyzed by flow cytometry. Endogenous and adoptive populations were distinguished by the allelic variants of CD45. B, OT-I T-cell expansion in peripheral blood expressed as the relative ratio between KO and WT cell counts on days 7 and 10 after BMDM injections (horizontal gray line represents the range of the initial NOS2 KO /WT ratio; N = 32–36). C, Percentage of CD62L-CD44+ cells (left) and CD8 and CD127 MFI (right) of WT, KO, and endogenous CD8 + T cells harvested from peripheral blood on day 7 ( N = 31). D, Recall response as determined by the ratio between WT and KO OT-I CD8 + T-cell infiltration in the spleen, lymph node, and liver 7 days after recall with SIINFEKL-pulsed BMDMs (+) or with PBS control (−). Horizontal gray line represents the range of the initial NOS2 KO /WT ratio ( N = 17–21). E, Recall response as determined by amount of CD44 + WT and NOS2 KO OT-I T cells per million CD45 + cells infiltrated in the spleen, lymph node, and liver 7 days after recall with BMDMs or PBS control ( N = 9–18). F, Flow cytometry analysis of CD44 in CD8 + T cells infiltrating the spleen, lymph node, and liver on day 37. All results [median ± interquartile range (IQR)] are pooled from three independent experiments and each data point represents an independent animal. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One sample t test relative to 1 ( B and D ), Tukey multiple comparisons paired test ( C – F ). #, P < 0.05; ##, P < 0.01; ###, P < 0.001; Unpaired t test.

Article Snippet: Mouse protein extracts (25–40 μg) were probed with primary antibodies against panNOS (CST #2977), NOS2 (CST #13120), and hypoxia-inducible factor (HIF)-1α (Novus NB-100–449) and detected using infrared-labeled donkey anti-rabbit (926–32213, LI-COR) and goat anti-mouse (926–68070, LI-COR) secondary antibodies in an Odyssey imaging system (LI-COR).

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry