Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Empagliflozin Protects against Pulmonary Ischemia/Reperfusion Injury via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent Mechanism.

doi: 10.1124/jpet.121.000956

Figure Lengend Snippet: Fig. 5. The effect of empagliflozin on protein phosphorylation after I/R injury. Left: Western blots of p-GSK-3b and t-GSK-3b (A), p-AKT and t-AKT (B), p-ERK1/2 and t-ERK1/2 (C), p-STAT-1 and t-STAT-1 (D), p-STAT-3 and t-STAT-3 (E), p-STAT-5 and t-STAT-5 (F) in lung tissues after I/R injury. Right: bar graph showing mean ratio of the phospho-/total band densities. **P < 0.01, ***P < 0.001, versus sham-operated mice; ###P < 0.001, versus control mice. n 5 5 per group.

Article Snippet: The primary antibodies used in this study were: p-glycogen synthase kinase-3b (GSK-3b), phosphorylated GSK-3b (Ser9); t-GSK-3b, total GSK-3b; p-protein kinase B (AKT), phosphorylated AKT (ser473); t-AKT, total AKT; pERK, phosphorylated ERK 1/2 (Thr202/Tyr204); t-ERK, total ERK1/2; p-signal transducer and activator of transcription (STAT)-1, phosphorylated STAT-1 (Tyr701); t-STAT-1, total STAT-1; P-STAT-3, phosphorylated STAT-3 (Tyr705); t-STAT-3, total STAT-3; p-STAT-5, phosphorylated STAT-5 (Tyr694); t-STAT-5, total STAT-5 (all primary antibodies, 1:1000, Cell Signaling, Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: The Journal of pharmacology and experimental therapeutics

Article Title: Empagliflozin Protects against Pulmonary Ischemia/Reperfusion Injury via an Extracellular Signal-Regulated Kinases 1 and 2-Dependent Mechanism.

doi: 10.1124/jpet.121.000956

Figure Lengend Snippet: Fig. 5. The effect of empagliflozin on protein phosphorylation after I/R injury. Left: Western blots of p-GSK-3b and t-GSK-3b (A), p-AKT and t-AKT (B), p-ERK1/2 and t-ERK1/2 (C), p-STAT-1 and t-STAT-1 (D), p-STAT-3 and t-STAT-3 (E), p-STAT-5 and t-STAT-5 (F) in lung tissues after I/R injury. Right: bar graph showing mean ratio of the phospho-/total band densities. **P < 0.01, ***P < 0.001, versus sham-operated mice; ###P < 0.001, versus control mice. n 5 5 per group.

Article Snippet: The primary antibodies used in this study were: p-glycogen synthase kinase-3b (GSK-3b), phosphorylated GSK-3b (Ser9); t-GSK-3b, total GSK-3b; p-protein kinase B (AKT), phosphorylated AKT (ser473); t-AKT, total AKT; pERK, phosphorylated ERK 1/2 (Thr202/Tyr204); t-ERK, total ERK1/2; p-signal transducer and activator of transcription (STAT)-1, phosphorylated STAT-1 (Tyr701); t-STAT-1, total STAT-1; P-STAT-3, phosphorylated STAT-3 (Tyr705); t-STAT-3, total STAT-3; p-STAT-5, phosphorylated STAT-5 (Tyr694); t-STAT-5, total STAT-5 (all primary antibodies, 1:1000, Cell Signaling, Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Csf2 Attenuated Sepsis-Induced Acute Kidney Injury by Promoting Alternative Macrophage Transition

doi: 10.3389/fimmu.2020.01415

Figure Lengend Snippet: Csf2 mediated alternative activation via the p-STAT5 pathway. (A) Representative immunofluorescence images of M1 macrophages showing the expression of HLA-DR (red, M1 marker) and CD206 (green, M2 marker) with Csf2 stimulation at different doses for 48 h. Slides were directly visualized using an Olympus fluorescence microscope at a 20X magnification, scale bar = 20 μm. (B) qRT-PCR was used to detect the mRNA levels of CD163 and IL-10 in M1 macrophages exposed to different doses of Csf2. Data are the mean ± SD of three separate experiments. The significance of differences was tested using Student's t -test. * p < 0.05. (C) Representative immunofluorescence images of M1 macrophages showing the expression of HLA-DR (red, M1 marker) and CD206 (green, M2 marker) after coculture with LPS-HK-2 for 4 days. Slides were directly visualized using an Olympus fluorescence microscope at a 20X magnification, scale bar = 20 μm. (D) Representative immunofluorescence images of M0 macrophages showing the expression of CD68 (red, M0 marker) and CD206 (green, M2 marker) following Csf2 stimulation at different doses for 48 h. Slides were directly visualized using an Olympus fluorescence microscope at a 20X magnification, scale bar = 20 μm. (E) Western blot analysis was used to detect CD206 (M2 marker), ARG1 (M2 marker), total STAT5 and phosphorylated STAT5 in M1 macrophages at the indicated times. Three independent experiments were performed.

Article Snippet: Membranes were incubated with primary Abs specific for the following proteins: Csf2 (Abcam, ab54429), CD206 (Proteintech, 18704-1-AP), ARG1 (Proteintech, 16001-1-AP), t-Stat5 (Abcam, ab227687), p-STAT5 (Cell Signaling Technology, mAb #4322), and Jak2 (Proteintech, 17670-1-AP).

Techniques: Activation Assay, Immunofluorescence, Expressing, Marker, Fluorescence, Microscopy, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Csf2 Attenuated Sepsis-Induced Acute Kidney Injury by Promoting Alternative Macrophage Transition

doi: 10.3389/fimmu.2020.01415

Figure Lengend Snippet: Blockade of Csf2 after CLP attenuated kidney macrophage alternative activation and slowed reparative tubule proliferation. (A) Serum and kidney tissues were collected for Scr and H&E staining at day 6. Representative immunofluorescence images of kidney tissue showing the expression of HLA-DR (M1 marker, red) and CD206 (M2 marker, green) after neutralizing Csf2 antibody or isotype antibody injection. Data are shown as the mean ± SD of 6 mice per group per experiment and were pooled from two independent experiments. (B–D) The indicated M2 markers (CD163 and IL10) and Csf2 were assessed using qRT-PCR. Data are shown as the mean ± SD of six mice per group per experiment, and two independent experiments were performed. * p < 0.05; two-tailed unpaired Student's t -test. (E) Western blotting was utilized to detect total STAT5 and phosphorylated STAT5 in the kidney cortex. Six mice were used per group per experiment, and two independent experiments were performed. (F,G) The concentrations of TNFα and IL10 in the kidney cortex were assayed by ELISA. Data are shown as the mean ± SD of 6 mice per group per experiment. (H) The apoptosis rate was analyzed in the kidneys of mice by TUNEL. Data are shown as the mean ± SD of 6 mice per group per experiment. (I) The collected kidneys from the four groups were stained with H&E. Scale bars: 20 μm. Kidney histology from CLP mice showing significant tubular epithelial swelling and brush border injury. Data are shown as the mean ± SD of six mice per group per experiment and were pooled from two independent experiments. (J) Photomicrograph of kidney tissue sections immunohistochemically stained for Ki67. Dense brown nuclear immunohistochemical staining indicates Ki67-positive status. Quantification of Ki67-positive tubular cells in the outer medulla. Data are shown as the mean ± SD of six mice per group per experiment and were pooled from two independent experiments, ** p < 0.01; two-tailed unpaired Student's t -test.

Article Snippet: Membranes were incubated with primary Abs specific for the following proteins: Csf2 (Abcam, ab54429), CD206 (Proteintech, 18704-1-AP), ARG1 (Proteintech, 16001-1-AP), t-Stat5 (Abcam, ab227687), p-STAT5 (Cell Signaling Technology, mAb #4322), and Jak2 (Proteintech, 17670-1-AP).

Techniques: Activation Assay, Staining, Immunofluorescence, Expressing, Marker, Injection, Quantitative RT-PCR, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Immunohistochemical staining

Journal: Frontiers in Immunology

Article Title: Csf2 Attenuated Sepsis-Induced Acute Kidney Injury by Promoting Alternative Macrophage Transition

doi: 10.3389/fimmu.2020.01415

Figure Lengend Snippet: Schematic representation of Csf2 regulation of macrophage transformation in sepsis-induced AKI. Csf2 secreted by tubular cells promotes Stat5 phosphorylation, reduces cell apoptosis, facilitates injured tubular cell repair and therefore attenuates kidney injury in vitro and in vivo .

Article Snippet: Membranes were incubated with primary Abs specific for the following proteins: Csf2 (Abcam, ab54429), CD206 (Proteintech, 18704-1-AP), ARG1 (Proteintech, 16001-1-AP), t-Stat5 (Abcam, ab227687), p-STAT5 (Cell Signaling Technology, mAb #4322), and Jak2 (Proteintech, 17670-1-AP).

Techniques: Transformation Assay, Phospho-proteomics, In Vitro, In Vivo