Figure 3.
Characterization of ATI2341 in vitro and in vivo. (a) 3D structure of the CXCR4 agonist pepducin ATI-2341 coupled to CXCR4. (b) p-pERK1/2 protein levels in WT BM neutrophils after stimulation with CXCR4 agonists. Blots are representative of n = 3 (CXCL12) and 2 (ATI2341) experiments. (c) Inhibition of CXCR2 signaling through CXCL12/CXCR4. WT neutrophils were preincubated with CXCR4 agonists and then allowed to migrate toward a CXCL1 gradient. The number of migrated cells was evaluated by flow cytometry; n = 3–6 mice per group. (d) Surface expression of CXCR4 in WT neutrophils after stimulation with CXCL12 or dose-dependent concentrations of ATI2341 (0.03, 0.3, 3, and 30 µM); n = 3 mice per group. (e) Viability of migrated neutrophils evaluated by flow cytometry from mice in c. (f) Experimental scheme to analyze neutrophil recruitment by IVM during TNF-α–induced inflammation. (g) Adhesion of neutrophils on cremasteric venules after treatment with TNF-α (inflammation) in vehicle- and ATI2341-treated neutrophils (left) and WT and Cxcr4+/1013 (WHIM) neutrophils (right, from transplant chimeras of WT and Cxcr4+/1013 mutant cells); n = 3–4 mice per group, normalized to vehicle or % of blood neutrophils in chimeric mice. (h) Infiltration efficiency of vehicle- and ATI2341-treated neutrophils (left) and WT and Cxcr4+/1013 (WHIM) neutrophils into the inflamed peritoneum after zymosan injection; n = 3–4 mice per group. (i) Peripheral blood counts in vehicle- and ATI2341-treated mice at ZT5; n = 7–8 mice per group. (j) O2 consumption (VO2), CO2 production (VCO2), energy expenditure (EE), respiratory quotient (RQ), general locomotor activity (activity), vertical activity (rearing), food intake (food), and drink intake (drink) of vehicle- and ATI2341-treated mice housed in metabolic cages for 3 days with food and water available ad libitum; n = 3–4 mice per group. AUs: arbitrary units. Data in c, e, and i are pooled from two experiments. Data in d, g, and h; right (Cxcr4+/1013) are from single experiments. Data in g and h; left (ATI) and j are representative of two experiments. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, as determined by one-way ANOVA (c–e), unpaired t test (i, g, and h; left), paired t test (g and h; right), and two-way ANOVA (j). IVM, intravital microscopy. Source data are available for this figure: SourceData F3.

Characterization of ATI2341 in vitro and in vivo. (a) 3D structure of the CXCR4 agonist pepducin ATI-2341 coupled to CXCR4. (b) p-pERK1/2 protein levels in WT BM neutrophils after stimulation with CXCR4 agonists. Blots are representative of n = 3 (CXCL12) and 2 (ATI2341) experiments. (c) Inhibition of CXCR2 signaling through CXCL12/CXCR4. WT neutrophils were preincubated with CXCR4 agonists and then allowed to migrate toward a CXCL1 gradient. The number of migrated cells was evaluated by flow cytometry; n = 3–6 mice per group. (d) Surface expression of CXCR4 in WT neutrophils after stimulation with CXCL12 or dose-dependent concentrations of ATI2341 (0.03, 0.3, 3, and 30 µM); n = 3 mice per group. (e) Viability of migrated neutrophils evaluated by flow cytometry from mice in c. (f) Experimental scheme to analyze neutrophil recruitment by IVM during TNF-α–induced inflammation. (g) Adhesion of neutrophils on cremasteric venules after treatment with TNF-α (inflammation) in vehicle- and ATI2341-treated neutrophils (left) and WT and Cxcr4+/1013 (WHIM) neutrophils (right, from transplant chimeras of WT and Cxcr4+/1013 mutant cells); n = 3–4 mice per group, normalized to vehicle or % of blood neutrophils in chimeric mice. (h) Infiltration efficiency of vehicle- and ATI2341-treated neutrophils (left) and WT and Cxcr4+/1013 (WHIM) neutrophils into the inflamed peritoneum after zymosan injection; n = 3–4 mice per group. (i) Peripheral blood counts in vehicle- and ATI2341-treated mice at ZT5; n = 7–8 mice per group. (j) O2 consumption (VO2), CO2 production (VCO2), energy expenditure (EE), respiratory quotient (RQ), general locomotor activity (activity), vertical activity (rearing), food intake (food), and drink intake (drink) of vehicle- and ATI2341-treated mice housed in metabolic cages for 3 days with food and water available ad libitum; n = 3–4 mice per group. AUs: arbitrary units. Data in c, e, and i are pooled from two experiments. Data in d, g, and h; right (Cxcr4+/1013) are from single experiments. Data in g and h; left (ATI) and j are representative of two experiments. Data are shown as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, as determined by one-way ANOVA (c–e), unpaired t test (i, g, and h; left), paired t test (g and h; right), and two-way ANOVA (j). IVM, intravital microscopy. Source data are available for this figure: SourceData F3.

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