Journal: International Journal of Molecular Sciences

Article Title: Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

doi: 10.3390/ijms22158042

Figure Lengend Snippet: Impact of IL-17A on MNs: ( A ) the MAP-2 immunostaining after different concentrations (5 ng/mL, 50 ng/mL, and 500 ng/mL) of IL-17A-stimulated MNs for three days; ( B ) the viability of MNs under the same IL-17A treatment for three days was evaluated using CCK-8 assay; ( C ) the MAP-2 positive MNs, counting manually in different IL-17A treatments; ( D ) the neurite length of MAP-2 positive MNs was calculated using Neuron J in the FIJI software; ( E ) the immunostaining of MAP-2 with the same IL-17A treatment at different times (three days and six days); ( F ) the MAP-2 positive MNs’ counting in IL-17A treatment at different times; ( G ) the length of the neurite was measured under the same treatment. * p < 0.05, ** p < 0.01, *** p < 0.001 versus controls, ns: no significance.

Article Snippet: Further, 0.5 µg/mL of IL-17A neutralizing antibody (1:1,000, BPS Bioscience, San Diego, CA, USA) and 1µg/mL of human IL17RA/IL-17R antibody pretreatment (1:500, R&D Systems, Minneapolis, MN, USA) for 0.5 h prior to adding IL-17A was performed for three days.

Techniques: Immunostaining, CCK-8 Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

doi: 10.3390/ijms22158042

Figure Lengend Snippet: Effect of IL-17F on MNs: ( A ) immunostaining of MAP-2 in IL-17F (5 ng/mL, 50 ng/mL, and 500 ng/mL) stimulated MNs; ( B ) the viability of MNs under different IL-17F treatments was measured using CCK-8 assay; ( C ) MAP-2 positive MNs counting under different IL-17F treatments; (D) the neurite length was calculated, using Neuron J in the FIJI software, under different treatment conditions in both cells, ns: no significance.

Article Snippet: Further, 0.5 µg/mL of IL-17A neutralizing antibody (1:1,000, BPS Bioscience, San Diego, CA, USA) and 1µg/mL of human IL17RA/IL-17R antibody pretreatment (1:500, R&D Systems, Minneapolis, MN, USA) for 0.5 h prior to adding IL-17A was performed for three days.

Techniques: Immunostaining, CCK-8 Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

doi: 10.3390/ijms22158042

Figure Lengend Snippet: Immunostaining of the IL-17 receptor (IL-17RA and IL-17RC) and MHCI in MNs: ( A ) immunostaining of IL-17RA, IL-17RC, and MHCI in FUS-WT-EGFP, FUS-P525L-EGFP, PBMCs, and a fibroblast: ( B ) co-staining of MAP-2 with IL-17RA, IL-17RC, and MHCI in FUS-WT-EGFP and FUS-P525L-EGFP cells; ( C ) the counting of IL-17RA positive cells in MNs; ( D ) IL-17RA positive staining in MNs; ( E ) MHCI positive staining in MNs. * p < 0.05 versus controls, ns: no significance.

Article Snippet: Further, 0.5 µg/mL of IL-17A neutralizing antibody (1:1,000, BPS Bioscience, San Diego, CA, USA) and 1µg/mL of human IL17RA/IL-17R antibody pretreatment (1:500, R&D Systems, Minneapolis, MN, USA) for 0.5 h prior to adding IL-17A was performed for three days.

Techniques: Immunostaining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Interleukin-17 and Th17 Lymphocytes Directly Impair Motoneuron Survival of Wildtype and FUS-ALS Mutant Human iPSCs

doi: 10.3390/ijms22158042

Figure Lengend Snippet: Antagonizing IL17 protects from IL17-induced neurodegeneration: ( A ) IL-17A-neutralizing pretreatment for 0.5 h, 50 ng/mL IL-17A stimulated the MNs for three days, MAP-2 immunostaining was performed after the treatment; ( B ) the viability of MNs under the same treatment mentioned before was calculated using CCK-8 assay; ( C ) MAP-2 positive MNs were counted and (D) the neurite length of MNs were measuredunder different treatments; ( E ) immunostaining of MAP-2 under anti-IL-17RA/IL-17R receptor pretreatment for 0.5 h, then 50 ng/mL IL-17A-stimulated for three days; ( F ) viability of MNs; ( G ) MAP-2 positive MNs counting; (H) neurite length of MAP-2 positive MNs using the aforementioned treatment.* p < 0.05, ** p < 0.01 versus controls, ns: no significance.

Article Snippet: Further, 0.5 µg/mL of IL-17A neutralizing antibody (1:1,000, BPS Bioscience, San Diego, CA, USA) and 1µg/mL of human IL17RA/IL-17R antibody pretreatment (1:500, R&D Systems, Minneapolis, MN, USA) for 0.5 h prior to adding IL-17A was performed for three days.

Techniques: Immunostaining, CCK-8 Assay

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: ( A ) Relative IL-8 mRNA expression in M2 macrophages treated with leptin with or without the inhibitors. M2 macrophages were pretreated with JAK inhibitor AG490 (50 μmol/L), PI3K inhibitor LY294002 (10 μmol/L), ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative IL-8 mRNA levels were analyzed by qRT-PCR. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01. ( B ) A representative image of Western blot for IL-8 protein expression in M2 macrophages treated in the same manner as (A). ( C ) A representative image of Western blot showing the time course of leptin-induced p38 and ERK 1/2 phosphorylation. M2 macrophages were treated with leptin (100 ng/mL) for 0–24 hours. ( D ) A representative image of Western blot showing leptin-induced ObR-dependent phosphorylation of ERK 1/2 and p38 and production of IL-8. M2 macrophages were pretreated with a polyclonal anti-human ObR antibody (4 μg/mL) for 16 hours and then treated with PBS or leptin (100 ng/mL). ( E ) Luciferase reporter assay to measure leptin-mediated IL-8 promoter activation. M2 macrophages were pretreated with ERK inhibitor PD980590 (10 μmol/L), p38 MAPK inhibitor SB203580 (20 μmol/L), or JNK inhibitor SP600125 (50 μmol/L) for 1 hour and then were treated with PBS or leptin (100 ng/mL). Relative luciferase units (RLU) normalized to β-galactosidase activity are shown. ***represents significant difference between the indicated groups versus the PBS group, P < 0.001. **represents significant difference between the indicated groups versus the Leptin + DMSO group, P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Luciferase, Reporter Assay, Activation Assay, Activity Assay

Journal: Oncotarget

Article Title: Leptin promotes migration and invasion of breast cancer cells by stimulating IL-8 production in M2 macrophages

doi: 10.18632/oncotarget.11761

Figure Lengend Snippet: The mouse xenograft model and leptin injection were conducted as in Figure . In the leptin-treated group, polyclonal rabbit anti-mouse IL-8 neutralization antibody (Abbexa Ltd, Cambridge, UK) was injected intraperitoneal in the mice at an initial dose of 0.2 mL per mouse and then 0.1 mL per mouse twice per week for 2 weeks. Polyclonal rabbit IgG (Bioss Ltd, Beijing, China) was used as the control. Mice received the injection of leptin and the antibodies for 2 weeks. ( A ) Tumor volume in the PBS, leptin, leptin + anti-IgG, and leptin + anti-IL8 groups. * P < 0.05. ( B ) Photos of tumor xenografts at day 21 after transplantation of human breast cancer cells. ( C ) Tumor wet weight at day 21 after the cell transplantation. ** P < 0.01. ( D ) Mouse survival. * P < 0.05. ( E ) Representative images of H & E staining of the lung tissue and the figure showing the number of metastatic nodules in the lung. ** P < 0.01. ( F ) Representative images of H & E staining of the liver tissue. ( G ) Representative images of IHC staining of CD68, Ki-67, and IL-8 in the tumor tissue and the figure showing quantitative analysis of the staining. ** P < 0.01.

Article Snippet: In the experiment of testing anti-mouse IL-8 antibody, mice in the leptin group were injected intraperitoneally with polyclonal rabbit anti-mouse IL-8 neutralizing antibody (Abbexa, Cambridge, UK) at an initial dose of 0.2 mL (45 μg/mL) per mouse and then 0.1 mL per mouse twice per week for 2 weeks and polyclonal rabbit IgG (Bioss) was used as the isotype control.

Techniques: Injection, Neutralization, Control, Transplantation Assay, Staining, Immunohistochemistry