Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: a The genes expression in immune system were analyzed by KEGG pathway enrichment analysis between chronic osteomyelitis-infected samples and non-infectious fractures samples. b Profiling of differentially expressed interferon-related genes between chronic osteomyelitis-infected samples and non-infectious fractures samples. c Representative images of immunohistochemical staining with monoclonal antibodies against IFN-λ1, CD80, CD86, and CD1a. d ELISA for IFN-λ1 concentrations from the serum of chronic osteomyelitis patients and non-infectious fracture patients. e Relative expression of IFN-λ1 on mRNA and protein level between LPS (100 ng/ml) pretreatment with dendritic cells and vehicle for 72 h. β-actin and GAPDH were used as an internal control. f Schematic representation of the experimental design of the establishment the co-culture between osteoclast and LPS-induced dendritic cells. g Representative images of TRAP staining of RAW264.7 cells treated with different groups. h Quantification of multinucleated TRAP-positive osteoclast number per well. The data in the figures represent the averages ± SD. Scale bars = 200 μm. Significant differences are indicated as * p < 0.05 or ** p < 0.01 paired using Student’s t -test unless otherwise specified.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Expressing, Infection, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: a Flow cytometry analysis of the apoptosis rate of RAW264.7 cells treated with RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 72 h with various doses of IFN-λ1. b Quantitative analysis of total apoptosis rate during osteoclastogenesis. c , d CCK-8 was performed in triplicate to analyze the cell viability of BMMs treated with varying doses of IFN-λ1 for 24 and 72 h with or without RANKL (100 ng/ ml) and M-CSF (50 ng/ml). The data in the figures represent the averages ± SD. N.S. represented as no significant difference. Significant differences are indicated as * p < 0.05 or ** p < 0.01 paired using Student’s t- test unless otherwise specified.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Flow Cytometry, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: a Representative images of FAK staining of RAW264.7 cells treated with RANKL and M-CSF alone or together with the indicated concentrations of IFN-λ1 treatment. F-actin using tetramethylrhodamine-conjugated phalloidin (red), focal contacts using anti-vinculin mAb, and nuclear counterstaining using DAPI (blue). Scale bar = 200 μm. Quantitative analysis of osteoclasts (nucleiå 3) and average osteoclast nuclei number in each field. b Representative images of FAK staining of RAW264.7 cells treated with RANKL and M-CSF alone or together with the indicated concentrations of IFN-λ1 treatment. F-actin using tetramethylrhodamine-conjugated phalloidin (red), focal contacts using anti-vinculin mAb, and nuclear counterstaining using DAPI (blue). Scale bar = 200 μm. Quantitative analysis of osteoclasts (nuclei > 3) and average osteoclast nuclei number in each field. The data in the figures represent the averages ± SD. Significant differences are indicated as * p < 0.05 or ** p < 0.01 paired using Student’s t- test unless otherwise specified.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Staining

Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: a , c , e Representative TRAP stain images of RAW264.7 cells and BMMs treated with RANKL or LPS-induced osteoclastogenesis. b , d , f Quantification of osteoclasts number per well. g RAW264.7 cells were plated on the bone slices and were cultured with RANKL or LPS for 6 days in the presence or absence of 100 ng/ml IFN-λ1. Scale bar = 200 μm. Quantification of the bone resorption area on the bone slices. h RAW264.7 cells were plated on the Osteo Assay Surface and were cultured with RANKL or LPS for 6 days in the presence or absence of 100 ng/ml IFN-λ1. Scale bar = 200 μm. Quantification of the bone resorption area on the bone slices. i BMMs were plated on the bone slices and were cultured with RANKL for 6 days in the presence or absence of 100 ng/ml IFN-λ1. Scale bar = 200 μm. Quantification of the bone resorption area on the bone slices. The data in the figures represent the averages ± SD. N.S. represented as no significant difference. Significant differences are indicated as * p < 0.05 or ** p < 0.01 paired using Student’s t- test unless otherwise specified.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Staining, Cell Culture

Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: a RAW264.7 cells were seeded in 96-well plates and treated with IFN-λ1 (100 ng/ml) for 24 h, followed by stimulation with 100 ng/ml RANKL and 50 ng/ml M-CSF. The intracellular location of the NFATc1 was observed by immunofluorescence staining using confocal microscopy. Scale bar = 800 μm. b The gray values of the Green and Blue staining were measured using the Image J software, and the mean values were plotted using excel. c Relative mRNA expression of CD9, c-Fos, Ctsk, PU.1, and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 24 h. d Relative expression of c-Fos and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 24 h in protein level. β-actin was used as an internal control. e Relative expression of CD9, MMP-9, CTSK, c-Fos, and NFATc1 during treatment with RANKL or LPS in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h in protein level. β-actin was used as an internal control. f Relative mRNA expression of mitf, c-Fos, Ctsk, CTR, and NFATc1 during treatment with RANKL in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h. g Relative mRNA expression of mitf, Ctsk, CTR, and OC-STAMP during treatment with LPS in the presence or absence of IFN-λ1 (100 ng/ml) for 72 h. h Relative mRNA expression of IL-1β, IL-6, and TNF-α during LPS-induced osteoclastogenesis in the presence or absence of IFN-λ1 (100 ng/ml). The data in the figures represent the averages ± SD. Significant differences are indicated as * p < 0.05 or ** p < 0.01 paired using Student’s t- test unless otherwise specified.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Software, Expressing

Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: a RAW264.7 cells were seeded in 96-well plates and treated with IFN-λ1 (100 ng/ml) for 60 min, followed by stimulation with 100 ng/ml RANKL and 50 ng/ml M-CSF. The intracellular location of the NF-κB p65 was observed by immunofluorescence staining using confocal microscopy. Scale bar = 800 μm. b Quantitative analysis of the percentage of positive cells (NF-ĸB p65 translocation from cytosol to nuclear) in all cells. c Quantitative analysis of the mean intensity of NF-ĸB p65 in the cells nuclear. d RAW264.7 cells were stimulated with RANKL with or without IFN-λ1 (100 ng/ml) for the 0–60 min. The cell lysates were analyzed using western blotting for p-NFκB p65, NF-κB p65, p-IκBα, and IκBα. β-actin was used as an internal control. e , f The expression of HMGB1, RAGE, and NLRP3 during osteoclastogenesis in the presence or absence of IFN-λ1 (100 ng/ml) on protein level. β-actin was used as an internal control. g , h Relative expression of HMGB1 and NLRP3 during osteoclastogenesis in the presence or absence of IFN-λ1 (100 ng/ml) on mRNA level. i RAW264.7 cells were stimulated with RANKL with or without IFN-λ1 (100 ng/ml) for the 0–60 min. The cell lysates were analyzed using western blotting for p-Jak1, Jak1, p-Tyk2, Tyk2, p-Stat1, Stat1, p-Stat2, and Stat2. β-actin was used as an internal control. j Representative images of immunohistochemical staining with monoclonal antibodies against p-p65, p-JAK1, p-STAT1, and p-STAT2. Scale bar = 200 μm. k Quantitative analysis of p-p65, p-JAK1, p-STAT1, and p-STAT2-positive cells per mm in each field. The data in the figures represent the averages ± SD. Significant differences are indicated as * p < 0.05 or ** p < 0.01 paired using Student’s t- test unless otherwise specified.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Immunofluorescence, Staining, Confocal Microscopy, Translocation Assay, Western Blot, Expressing, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Dendritic cells-derived interferon-λ1 ameliorated inflammatory bone destruction through inhibiting osteoclastogenesis

doi: 10.1038/s41419-020-2612-z

Figure Lengend Snippet: Schematic diagrams showing the potential mechanism in the protective effects of dendritic cells derived IFN-λ1 on inflammatory bone destruction through inhibiting osteoclastogenesis.

Article Snippet: Human IFN-λ1 ELISA Kit was obtained from Bioss (Beijing, China).

Techniques: Derivative Assay

Journal: Translational Cancer Research

Article Title: DNMT3A promotes glioma growth and malignancy via TNF-α/NF-κB signaling pathway

doi: 10.21037/tcr-23-1943

Figure Lengend Snippet: Expression characteristics of DNMT3A gene. (A) The protein expression level of DNMT3A in four cell lines (SVG, BT142, SW1783, and SW1088), with GAPDH as the internal reference. (B) The mRNA expression level of DNMT3A in four cell lines (SVG, BT142, SW1783, and SW1088). (C) Verification of the protein knockdown effect of DNMT3A in two groups of sh-DNMT3A SW1088 cell lines, with GAPDH as the internal reference. (D) The mRNA expression level of DNMT3A in two groups of sh- DNMT3A SW1088 cell lines. (E) Immunohistochemistry of DNMT3A in clinical samples revealed the expression characteristics of DNMT3A. The staining method is shown in the “Methods” section. (F) The expression of DNMT3A was correlated with GFAP expression in glioma samples by immunohistofluorescence assay. (G) Detect the difference in TNF-α secretion after silencing DNMT3A using ELISA. (H) The phosphorylation of p65 can be inhibited by silencing the expression of DNMT3A and can be reversed by adding exogenous TNF-α. **, P<0.01; ***, P<0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA; sh, short hairpin; NC, negative control; NBT, normal brain tissue; DAPI, 4',6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; TNF-α, tumor necrosis factor-α; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: TNF-α levels in the culture supernatants of SW1088 cells were analyzed using the ELISA assay kit (Cat. No. #BSKH1014; Bioss Company, Beijing, China).

Techniques: Expressing, Immunohistochemistry, Staining, Immunohistofluorescence, Enzyme-linked Immunosorbent Assay, Negative Control