Journal: PLoS pathogens

Article Title: Myeloid derived hypoxia inducible factor 1-alpha is required for protection against pulmonary Aspergillus fumigatus infection.

doi: 10.1371/journal.ppat.1004378

Figure Lengend Snippet: Figure 6. Decreased neutrophil levels in HIFC mice correlates to decreased production of CXCL1 and not migration defects. Migration of littermate (WT) and HIFC BMDNs in the presence of (A) RPMI media containing FBS or nothing or (B) 12 hr BALF from HIFC or WT mice inoculated with 76107 conidia (Af) was measured using a 3 mm transwell chamber. (C) In vivo CXCL1 protein was measured in BALFs from 4 and 8 hr infections using an ELISA from AssayBiotech. (D) WT and HIFC BMDMs were incubated with A. fumigatus conidia in a 10:1 ratio for 8 hrs. mRNA abundance of cxcl1 was determined using quantitative RT-PCR, normalized to rpl13a, and relative to the HIFC sample (3 biological and 3 technical replicates). (E) In vivo CXCL2 and CXCL5 protein was measured in BALFs from 8 hr infections. (F) In vivo cytokine protein production was measured in BALFs from the same model in (B) using a cytokine luminex bead array from BioRad. (C,E) Data represent 3–4 biological replicates and depicted as mean plus SEM (N.D. = not detectable). * indicates a P value of ,0.03 and ** indicates a P value of ,0.05 (unpaired Students t test). doi:10.1371/journal.ppat.1004378.g006

Article Snippet: Commercially available ELISA kits for CXCL1 (Assay Biotech, OK-0189), CXCL2 (R&D systems, DY452), and CXCL5 (R&D Systems, DY443) were used according to the manufactures’ instructions.

Techniques: Migration, In Vivo, Enzyme-linked Immunosorbent Assay, Incubation, Quantitative RT-PCR, Luminex

Journal: The Journal of Immunology Author Choice

Article Title: Macrophagic Extracellular Vesicle CXCL2 Recruits and Activates the Neutrophil CXCR2/PKC/NOX4 Axis in Sepsis

doi: 10.4049/jimmunol.2100229

Figure Lengend Snippet: Chemokine profile of the control and LPS-induced Raw264.7 EVs. (A) The chemokine profile of EVs from control and LPS-induced Raw264.7 cells. (B) The CXCL2 level (mean ± SD) in the control-Raw-EV and LPS-Raw-EV groups. The experiments were repeated three times. **p < 0.01 versus the control-Raw264.7-EV group. (C) PKH67-labeled control-Raw264.7-EVs and LPS-Raw264.7-EVs were injected into wild-type C57BL/6 mice (n = 6 per group) via the tail vein. The colocalization of PKH67-EVs and CXCL2 in the liver was detected by immunofluorescence (original magnification × 200). (D and E) GO analysis and KEGG analysis of various chemokines expressed in EVs from control and LPS-induced Raw264.7 cells.

Article Snippet: The quantification of CXCL2 (KE10022; Proteintech), CXCL7 (DY393; R&D Systems, Minneapolis, MN), CXCL17 (AD7670Hu; Andy Gene, Beijing, China), CCL2 (KE00091; Proteintech), CCL3 (KE00092; Proteintech), and CCL5 (KE00093; Proteintech) was performed using quantitative ELISA.

Techniques: Control, Labeling, Injection, Immunofluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Macrophagic Extracellular Vesicle CXCL2 Recruits and Activates the Neutrophil CXCR2/PKC/NOX4 Axis in Sepsis

doi: 10.4049/jimmunol.2100229

Figure Lengend Snippet: LPS-induced CXCL2 of Raw264.7 EVs attracts neutrophils in vitro and in vivo and activates neutrophils via the CXCR2/PKC/NOX4 pathway. (A) The CXCL2 level (mean ± SD) of Raw264.7 in the control, LPS, LPS + lenti-CXCL2, and LPS + siRNA-CXCL2 groups detected by Western blotting (WB). The experiments were repeated three times. *p < 0.01 versus the control group, &p < 0.01 versus the LPS group, #p < 0.01 versus the LPS group. (B) The CXCL2 level (mean ± SD) in Raw264.7 EVs of the control, LPS, LPS + lenti-CXCL2, and LPS + siRNA-CXCL2 groups detected by WB. The experiments were repeated three times. (C) Raw264.7 EVs derived from the control, LPS, LPS + lenti-CXCL2, and LPS + siRNA-CXCL2 groups induced BMDN chemotaxis (mean ± SD) (n = 5 fields per group). (D) The expression of CXCR2, PKC, and NOX4 (mean ± SD) of BMDNs in the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EVs groups was detected by WB. The experiments were repeated three times. (E) H&E staining, MPO+ cells, and Ly-6G+ cells (mean ± SD) in the liver of the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EV group mice (n = 6 per group) detected by immunohistochemistry. Scale bars, 100 μm. (F–H) ALT, AST, and LDH levels (mean ± SD) in the serum of the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EV group mice (n = 6 per group). (I) The number of systemic circulating neutrophils (mean ± SD) in wild-type C57BL/6 mice treated with control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EVs (n = 3 per group). (J) The expression of CXCR2, PKC, and NOX4 (mean ± SD) in the liver of the control EVs, LPS-EVs, (LPS + lenti-CXCL2)-EVs, and (LPS + siRNA-CXCL2)-EV group mice was detected by WB. The experiments were repeated three times. *p < 0.01 versus the control-Raw264.7-EV group, &p < 0.01 versus the LPS-Raw264.7-EV group, #p < 0.01 versus the LPS-Raw264.7-EV group.

Article Snippet: The quantification of CXCL2 (KE10022; Proteintech), CXCL7 (DY393; R&D Systems, Minneapolis, MN), CXCL17 (AD7670Hu; Andy Gene, Beijing, China), CCL2 (KE00091; Proteintech), CCL3 (KE00092; Proteintech), and CCL5 (KE00093; Proteintech) was performed using quantitative ELISA.

Techniques: In Vitro, In Vivo, Control, Western Blot, Derivative Assay, Chemotaxis Assay, Expressing, Staining, Immunohistochemistry

Journal: The Journal of Immunology Author Choice

Article Title: Macrophagic Extracellular Vesicle CXCL2 Recruits and Activates the Neutrophil CXCR2/PKC/NOX4 Axis in Sepsis

doi: 10.4049/jimmunol.2100229

Figure Lengend Snippet: EVs from the serum of patients with sepsis attract neutrophils and activate the neutrophil CXCR2/PKC/NOX4 pathway in vitro. (A) The chemokine profile of EVs from the serum of healthy individuals and patients with sepsis. (B–G) CXCL2, CXCL7, CXCL17, CCL2, CCL3, and CCL5 levels (mean ± SD) in EVs from the serum of healthy individuals (n = 8) and patients with sepsis (n = 26) detected using ELISA. *p < 0.05 versus the healthy-EV group, ***p < 0.01 versus the healthy-EV group. (H) sEVs derived from healthy control (HC) and patients with sepsis-induced PMN chemotaxis (mean ± SD) (n = 5 fields per group). (I) The expression of CXCR2, PKC, and NOX4 (mean ± SD) in the PMNs of the HC-sEV– and sepsis-sEV–treated groups was detected by Western blotting. The experiments were repeated three times. **p < 0.01 versus the healthy-EV group, #p < 0.05 versus the healthy-EV group, &p < 0.05 versus the healthy-EV group.

Article Snippet: The quantification of CXCL2 (KE10022; Proteintech), CXCL7 (DY393; R&D Systems, Minneapolis, MN), CXCL17 (AD7670Hu; Andy Gene, Beijing, China), CCL2 (KE00091; Proteintech), CCL3 (KE00092; Proteintech), and CCL5 (KE00093; Proteintech) was performed using quantitative ELISA.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Chemotaxis Assay, Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: PDCD10 promotes the aggressive behaviors of pituitary adenomas by up-regulating CXCR2 and activating downstream AKT/ERK signaling

doi: 10.18632/aging.204206

Figure Lengend Snippet: Activation of CXCR2 rescues the inactivation of AKT/ERK signaling and the tumor-suppressive effects induced by PDCD10 silencing. ( A , B ) Western blotting was performed to examine the phosphorylation levels of AKT and ERK1/2 after recombinant CXCL2 administration (200ng/ml) in Att-20 and TtT/GF cells with PDCD10 silencing. ( C , D ) CCK-8 assay was used to evaluate cell proliferation potential after CXCL2 administration in Att-20 and TtT/GF cells with PDCD10 silencing. ( E , F ) Relative migration rate of ATT-20 and TtT/GF cells with PDCD10 silencing was analyzed by scratch assay after CXCL2 administration (magnification: 100x). ( G , H ) Transwell invasion assay was employed to assess the invasion capacity of Att-20 and TtT/GF cells with PDCD10 silencing after CXCL2 administration (magnification: 200x). * P < 0.05.

Article Snippet: Recombinant mouse CXCL2 protein (R&D System) was added into the culture medium to study the signaling pathways in a subset of experiments as indicated.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Recombinant, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Invasion Assay