Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: The development of immune cells in bone marrow and spleen were not affected due to DCAF1 deficiency in macrophages. ( A ) The genotype of DCAF1 myeloid cell-conditional KO mice was determined by PCR analysis. ( B ) The expressions of DCAF1 in the T cells and macrophages of DCAF1 fl/fl Lyz2 cre/+ mice were determined by Immunoblotting analysis. ( C–D ) The populations of B lymphocytes, T lymphocytes, macrophages, and neutrophils in the spleen from DCAF1 fl/fl Lyz2 +/+ (WT) or DCAF1 fl/fl Lyz2 cre/+ mice were analyzed by Flow cytometer. Representative FACS plots were shown. In this study, the data were shown as the mean ± SEM. The data were acquired from three independent experiments. The significance of data in ( C–D) was calculated by Student’s t -test. ns indicates no statistical significance.

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Western Blot, Flow Cytometry

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: Pro-inflammatory cytokines were elevated in macrophages with DCAF deficiency. ( A–B ) The mRNA and protein levels of pro-inflammatory cytokines in primary BMDMs induced by LPS were determined by qRT-PCR and specific ELISAs, respectively. ( C ) The expressions of osteoclasts-specific marker genes were detected by qPCR. ( D ) The efficiency of osteoclast generation from WT and DACF1-deficient BMDMs were measured by TRAP staining. ( E ) The mRNA levels of pro-inflammatory cytokines in WT and DCAF1-deficient osteoclasts induced by LPS were determined by qRT-PCR. The data were presented as fold change as compared to the internal control. In this study, the data were presented as the mean ± SEM. The significances of differences comparisons between the responses group in the two genotypes were determined by a two-tailed Student’s t -test. Three independent experiments were operated to acquire the results. * P < 0.05; ** P < 0.01.

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Quantitative RT-PCR, Marker, Staining, Control, Two Tailed Test

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: DCAF1 deficiency in macrophages induced severe symptoms in S. aureus -induced osteomyelitis. ( A ) The body weight was monitored every two days after the mice were injected with S. aureus (10 6 CFU) (n = 10). ( B ) After the injection of S. aureus , bacterial burdens were enumerated from the infected femurs and liver on day 14 (n=10). ( C ) The cortical bone loss (mm 3 ) and reactive bone formation (mm 3 ) were determined by μCT three-dimensional analysis. ( D ) After the injection of S. aureus , inflammatory tissues were collected and the mRNA levels of inflammatory cytokines were determined by qRT-PCR on day 8. In this study, the data were shown as the mean ± SEM. ( E ) Peripheral blood was collected and the secretion of these inflammatory cytokines were determined by ELISA on day 8. ( F ) The expressions of RankL and Opg inflammatory tissues were detected by qPCR. Three independent experiments were operated to acquire the results. The significance of data in ( A–E ) was calculated by Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001..

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Injection, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Inflammation Research

Article Title: Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus -Induced Osteomyelitis

doi: 10.2147/JIR.S307316

Figure Lengend Snippet: DCAF1 deficiency induced p38 hyperactivation. ( A–B ) BMDMs were isolated from WT or DCAF1 fl/fl Lyz2 cre/+ mice. Next, LPS was applied to stimulate BMDMs. The phosphorylation and total proteins of NF-kB and MAPKs signal-related markers were determined by Immunoblotting analysis. ( C ) BMDMs were pre-treated with p38 inhibitor (Doramapimod,100 nM) for 2 h prior to incubating with LPS. Next, the mRNA levels of inflammatory cytokines were determined by qRT-PCR. In this study, the data were presented as the mean ± SEM. The data were acquired from three independent experiments. The significance of data in ( C ) was calculated by Student’s t -test. * P < 0.05.

Article Snippet: The primary antibody against DCAF1 was purchased from Abcam (ab1766, Shanghai, China).

Techniques: Isolation, Phospho-proteomics, Western Blot, Quantitative RT-PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: Maackiain dampens osteoclastogenesis via attenuating RANKL‐stimulated NF‐κB signalling pathway and NFATc1 activity

doi: 10.1111/jcmm.15647

Figure Lengend Snippet: Maackiain suppressed the activation of NF‐κB signalling pathway. BMMs were stimulated with RANKL for various periods with or without 40 μmol/L Maackiain. The total protein was examined by Western blot. It indicated that Maackiain could inhibit the degradation of IκB‐α on the time‐points of 10 and 20 min, and dampened the phosphorylation of p65 since the time‐point of 20 min. n = 3; ** P < .01, *** P < .001

Article Snippet: Primary antibody against p‐p65 (Cat#3031S) was obtained from Cell Signalling Technology.

Techniques: Activation Assay, Western Blot, Phospho-proteomics

Journal: Scientific Reports

Article Title: ESS2 controls prostate cancer progression through recruitment of chromodomain helicase DNA binding protein 1

doi: 10.1038/s41598-023-39626-0

Figure Lengend Snippet: ESS2 regulated NF-κB/CHD1 target genes. ( a ) Heatmap data of prostate cancer-related genes by microarray analysis in PC3-shC and PC3-shESS2 cells. ( b ) RT-qPCR of TMPRSS2 in PC3-shC and PC3-shESS2 cells normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. ( c ) Heatmap data of NF-κB/CHD1 target genes by microarray analysis in PC3-shC and PC3-shESS2 cells. ( d ) Fold changes in gene expression levels (PC3-shESS2/PC3-shC) in ( c ). ( e ) RT-qPCR of TNF and IL6 in PC3-shC and PC3-shESS2 cells normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. ( f ) Western blotting of p65, CHD1, and β-actin in PC3-shC and PC3-shESS2 cells. Raw data was shown in Supplementary Fig. . ( g ) ChIP-qPCR analysis of TNF at the TSS site (TSS) and 3′-UTR site (3′-UTR) with anti-p65, anti-CHD1, and anti-histoneH3K36 trimethylation antibodies in PC3-shC and PC3-shESS2 cells normalized to the input level. *, p < 0.05, Student’s t-test. ( h ) Luciferase reporter assays using a p65/p50 expression vectors and/or recombinant TNFα to activate NF-κB on NF-κB response element (NFκRE)-luc reporter vector in HEK293 cells. ( i ) Luciferase reporter assays using a NFAT expression vector to activate NFAT on NFAT response element (NFAT RE)-luc reporter vector in HEK293 cells. ( j ) Luciferase reporter assays using 1 μM phorbol 12-myristate 13-acetate (PMA) to activate AP-1 on AP1 response element (AP1 RE)-luc reporter vector in HEK293 cells. ( k ) ESS2 knockdown by esiRNA decreased the transcriptional activities of NFAT in HEK293 cells. ( l ) RT-qPCR of ESS2 in PC3-shC and PC3-shESS2 cells treated with/without 1 μM ionomycin for 24 h, normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. ( m ) Scatter plots showing correlations between ESS2 and NF-κB/CHD1 target gene expression ( IER3 , LIF , and CSF2 ) in patients with prostate cancer. For RT-qPCR, ChIP-qPCR and luciferase reporter assays, each experiment was performed at least three times, and the results are presented as means ± standard deviations.

Article Snippet: For western blotting and/or ChIP, we used antibodies against p65 (cat. no. 6956 [Cell Signaling Technology, Danvers, MA, USA]; cat. no. ab16502 [Abcam, Cambridge, UK]), PPAR-γ (cat. no. PP-A3409A-00; Perseus Proteomics), VDR (cat. no. sc-13133; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ESS2/DGCR14 (cat. no. HPA001222; Sigma), CHD1 (cat. no. sc-271626; Santa Cruz Biotechnology), p21/CDKN1A (cat. no. sc-817; Santa Cruz Biotechnology), p27/CDKN1B (cat. no. sc-1641; Santa Cruz Biotechnology), GAPDH (cat. no. 10494–1-AP; Protein Group) and β-actin (cat. no. sc-47778; Santa Cruz Biotechnology).

Techniques: Microarray, Quantitative RT-PCR, Gene Expression, Western Blot, ChIP-qPCR, Luciferase, Expressing, Recombinant, Plasmid Preparation, Knockdown, esiRNA, Targeted Gene Expression

Journal: Scientific Reports

Article Title: ESS2 controls prostate cancer progression through recruitment of chromodomain helicase DNA binding protein 1

doi: 10.1038/s41598-023-39626-0

Figure Lengend Snippet: ESS2 regulated CCL2 , WNT5A , and TGFB1 mRNA levels. ( a ) Heatmap data of CCL mRNAs by microarray in PC3-shC and PC3-shESS2 cells. ( b ) RT-qPCR of CLL2 mRNA in PC3-shC and PC3-shESS2 cells normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. ( c ) ChIP-qPCR analysis of the CCL2 gene at the TSS site (TSS) and 3′-UTR site (3′-UTR) with anti-p65, anti-CHD1, and anti-histoneH3K36 trimethylation antibodies in PC3-shC and PC3-shESS2 cells normalized to the input level. *, p < 0.05, Student’s t-test. ( d ) RT-qPCR of CCL2 in siC- or siESS2-transfected LNCaP cells normalized to the level of GAPDH mRNA. *, p < 0.05, Student’s t-test. ( e ) Left panel: heatmap data for WNT-related genes in PC3-shC and PC3-shESS2 cells. Right panel: fold changes in gene expression levels (PC3shESS2/PC3-shC) in microarray data. ( f ) RT-qPCR of WNT5A in PC3-shC and PC3-shESS2 cells. *, p < 0.05, Student’s t-test. ( g ) Scatter plots showing correlations of ESS2 and WNT5A expression in patients with prostate cancer. ( h ) RT-qPCR of TGFB1 in PC3-shC and PC3-shESS2 cells. ( i ) Scatter plots showing correlations of ESS2 and TGFB1 expression in patients with prostate cancer. ( j ) RT-qPCR of MMP9 mRNA in PC3-shC and PC3-shESS2 cells with/without TGFβ normalized to the level of GAPDH mRNA. ( k ) Luciferase reporter assays using a constitutively active TGFβR1 (CA-TGFβR) expression vector to activate SMAD2/3 on Smad binding element (SBE)-luc reporter vector in HEK293 cells. ( l ) ESS2 knockdown by esiRNA decreased the transcriptional activities of SMAD2/ in HEK293 cells. *, p < 0.05, Student’s t-test. ( m ) RT-qPCR of CHD1 , TNF , TGFB1, VDR and PPARG in PC3 cells transfected with control siRNA (siC) or CHD1 siRNA(siCHD1) , normalized to the levels of GAPDH mRNA. *, p < 0.05, Student’s t-test. For RT-qPCR, ChIP-qPCR and luciferase reporter assays, each experiment was performed at least three times, and the results are presented as means ± standard deviations.

Article Snippet: For western blotting and/or ChIP, we used antibodies against p65 (cat. no. 6956 [Cell Signaling Technology, Danvers, MA, USA]; cat. no. ab16502 [Abcam, Cambridge, UK]), PPAR-γ (cat. no. PP-A3409A-00; Perseus Proteomics), VDR (cat. no. sc-13133; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ESS2/DGCR14 (cat. no. HPA001222; Sigma), CHD1 (cat. no. sc-271626; Santa Cruz Biotechnology), p21/CDKN1A (cat. no. sc-817; Santa Cruz Biotechnology), p27/CDKN1B (cat. no. sc-1641; Santa Cruz Biotechnology), GAPDH (cat. no. 10494–1-AP; Protein Group) and β-actin (cat. no. sc-47778; Santa Cruz Biotechnology).

Techniques: Microarray, Quantitative RT-PCR, ChIP-qPCR, Transfection, Gene Expression, Expressing, Luciferase, Plasmid Preparation, Binding Assay, Knockdown, esiRNA, Control

Journal: Drug Design, Development and Therapy

Article Title: Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway

doi: 10.2147/DDDT.S361748

Figure Lengend Snippet: Ginsenoside Rb1 inhibits NLRP3 inflammasome activation and the NF-κB pathway in BLM-induced pulmonary inflammation in mice on day 3. ( A ) The expression of NLRP3, ASC, caspase-1, caspase-1 p10, and pro IL-1β in lung tissues, measured using Western blotting (n=6). ( C ) Immunofluorescence co-localization of NLRP3 and CD11c. ( D ) The expression of phosphorylated IκBα and NF-κB p65 in lung tissues, measured using Western blotting (n=6). ( B ) ( E ) Quantitative analysis of the Western blots shown in ( A ) and ( D ). (n=6, *P < 0.05, **P< 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: Antibodies against NF-κB p65 (10745–1), IκBα (10268–1), α-smooth muscle actin (α-SMA; 80008–1), collagen type I (Col I) (66761–1), and CD11c (17342–1), as well as secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence

Journal: Drug Design, Development and Therapy

Article Title: Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway

doi: 10.2147/DDDT.S361748

Figure Lengend Snippet: G-Rb1 inhibits BLM-induced NLRP3 inflammasome activation and the NF-κB pathway in lungs on day 21. ( A ) Representative Western blots of NLRP3, ASC, caspase-1, caspase-1 p10, and pro IL-1β expression in lung tissues (n=6). ( C ) Representative Western blots of IκBα and NF-κB p65 phosphorylation in lung tissues (n=6). ( B ) ( D ) Quantitative analysis of the Western blots shown in A and C. (n=6, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Antibodies against NF-κB p65 (10745–1), IκBα (10268–1), α-smooth muscle actin (α-SMA; 80008–1), collagen type I (Col I) (66761–1), and CD11c (17342–1), as well as secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Activation Assay, Western Blot, Expressing, Phospho-proteomics

Journal: Drug Design, Development and Therapy

Article Title: Ginsenoside Rb1 Alleviates Bleomycin-Induced Pulmonary Inflammation and Fibrosis by Suppressing Central Nucleotide-Binding Oligomerization-, Leucine-Rich Repeat-, and Pyrin Domains-Containing Protein Three Inflammasome Activation and the NF-κB Pathway

doi: 10.2147/DDDT.S361748

Figure Lengend Snippet: G-Rb1 suppresses the NF-κB pathway in macrophages whereas MCC950 has no inhibitory effect on the NF-κB pathway. ( A ) ( D ) Representative Western blots of pIκBα/IκBα and pNF-κB p65/NF-κB p65. ( B ) ( E ) Quantitative analysis of the Western blots shown in ( A ) and ( D ). ( C ) Nuclear translocation of NF-κB p65 in macrophages examined using immunofluorescence. (n≥3, *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Antibodies against NF-κB p65 (10745–1), IκBα (10268–1), α-smooth muscle actin (α-SMA; 80008–1), collagen type I (Col I) (66761–1), and CD11c (17342–1), as well as secondary antibodies were purchased from Proteintech (Rosemont, IL, USA).

Techniques: Western Blot, Translocation Assay, Immunofluorescence