Journal: Wound Repair and Regeneration

Article Title: Activation of latent transforming growth factor-β1 by nitric oxide in macrophages: Role of soluble guanylate cyclase and MAP kinases

doi: 10.1111/j.1524-475x.2009.00509.x

Figure Lengend Snippet: Figure 2. Nitric oxide induces a dose-dependent increase in ac- tive and latent transforming growth factor-b1 (TGF-b1) protein expression in RAW 264.7 macrophage-like cells. (A) RAW 264.7 cells were treated with 10–1,000 mM S-nitroso-N-acetyl-D,L-pen- icillamine (SNAP) for 24 hours, and the expression of TGF-b1 was analyzed as described in ‘‘Materials and methods.’’ Re- sults shown are the mean SEM from six to seven indepen- dent experiments (np < 0.005 vs. control; nnp < 0.05 vs. 10 mM SNAP). (B) RAW 264.7 cells were treated with 100 mM SNAP or decomposed SNAP (either by heat treatment [T] or pH change) for 24 hours, and the expression of TGF-b1 was analyzed as described in ‘‘Materials and methods.’’ Results shown are the mean SEM. from four independent experiments (np < 0.05 vs. control and decomposed SNAP (pH), nnp < 0.01 vs. decom- posed SNAP (T), #p < 0.05 vs. control and decomposed SNAP (T), ##p < 0.01 vs. decomposed SNAP (pH). (C) RAW 264.7 cells were untreated, treated with 100 mM SNAP, or 100 mM decomposed SNAP. Cell lysates or defined amounts (5, 50, or 100 ng) of recombinant human latency-associated peptide (LAP) were subjected to anti-LAP and b-actin Western blot as described in ‘‘Materials and methods.’’ The experiment is rep- resentative of three; numbers indicate quantification (mean - SEM) of LAP expression normalized for b-actin expression.

Article Snippet: Antibodies to human TGF-b1 and human LAP antibody were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Control, Recombinant, Western Blot

Journal: Cancer Medicine

Article Title: The expression and role of SUZ12 in lung adenocarcinoma

doi: 10.1002/cam4.70190

Figure Lengend Snippet: The effect of SUZ12 on metastasis related genes expression was tested by qRT‐PCR and western blotting. sh‐SUZ12 increased MMP1/2/3/9 mRNA expression (A) and TIMP1/2 protein expression (B), while decreased MMP14 mRNA expression (A), MMP1/9/14 (B), TIMP3 (B), and ITGB1/5(F) protein expression, without significantly effected ITGBs mRNA expression (E). oe‐SUZ12 increased MMP14 mRNA (C) and protein (D) expression, while decreased TIMP2 mRNA expression (C) and TIMP1/2 (D) protein expression.

Article Snippet: The list of primary antibodies: SUZ12 (1 μg/mL, Abcam Cambridge, cat no: ab12073), CDK2 (1:1000; Proteintech, USA, cat. no. 10122‐1‐AP), CDK3 (1:2000; Proteintech, USA, cat. no. 55103‐1‐AP), CDK6 (1:2000; Proteintech, USA, cat. no. 14052‐1‐AP), cyclin D1 (1:5000; Proteintech, USA, cat. no. 26939‐1‐AP), cyclin E1 (1:1000; Proteintech, USA, cat. no. 11554‐1‐AP), p18 (1:1000, BOSTER China, cat. no. M03299‐1), p19 (1:1000, BOSTER China, cat. no. MA1075), p53 (1:5000; Proteintech, USA, cat. no. 60283‐2‐Ig), p‐p53 (1:2000; Proteintech, USA, cat. no. 28961‐1‐AP), p57 (1:1000, BOSTER China, cat. no. BM4129), Rb (1:1000, BOSTER China, cat. no. BM4500), pRb (1:1000, BOSTER China, cat. no. BM4338), Bcl‐2 (1:1000; Proteintech, USA, cat. no. 26593‐1‐AP), Bax (1:2000; Proteintech, USA, cat. no. 50599‐2‐lg), E‐cadherin (1:5000; Proteintech, USA, cat. no. 20874‐1‐AP), N‐cadherin (1:3000; Proteintech, USA, cat. no. 22018‐1‐AP), vimentin (1:4000; Proteintech, USA, cat. no. 10366‐1‐AP), MMP1 (1:1000, BOSTER China, cat. no. A00733‐1), MMP2 (1:500, BOSTER China, cat. no. BM4075), MMP9 (1:1000, BOSTER China, cat. no. PB0709), MMP14 (1:1000, BOSTER China, cat. no. BM4119), TIMP1 (1:1000, Bioss China, cat. no. bs‐0415R), TIMP2 (1:1000, Bioss China, cat. no. bs‐10395R), TIMP3 (1:1000; Proteintech, USA, cat. no. 10858‐1‐AP), ITGB1 (1:1000, BOSTER China, cat. no. BM4308), ITGB3 (1:1000, BOSTER China, cat. no. BA1670), ITGB5 (1:1000, BOSTER China, cat. no. A04201‐1), nm23 (1:1000, BOSTER China, cat. no. BA3787), PD‐L1 (1:3000; Proteintech, USA, cat. no. 66248‐1‐Ig), and β‐actin (1:5000; Proteintech, USA, cat. no. 66009‐1‐Ig).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Neuroinflammation

Article Title: Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

doi: 10.1186/s12974-024-03119-2

Figure Lengend Snippet: Monocyte-specific overexpression of human cystatin F exacerbated cognitive impairment in APP/PS1 transgenic mice. A Construction of monocyte-specific overexpression of human cystatin F under the control of the human CD68 promoter. B Schematic for generating APP/PS1/Hmo-cys F + mice, behavioural tests, and pathological analysis. C Identification of transgenic mice by PCR analysis of the genomic DNA. D Transcript levels of human cystatin F in the monocytes of 6-month-old WT (n = 6), Hmo-cys F + (n = 6), APP/PS1 (n = 6), and APP/PS1/Hmo-cys F + mice (n = 6). E Immunofluorescence and statistical analysis of Aβ plaques in brain sections from transgenic mice. Scale bar: 1000 μm F , G ELISA analysis for soluble F Aβ40 and G Aβ42 levels in brains extracted with RIPA buffer. H , I ELISA analysis for insoluble H Aβ40 and I Aβ42 levels in brains extracted with SDS buffer. J , K MWM analysis showing the latency (s), L number of target crosses, and M mean speed (cm/s) in the invisible platform test. N Representative images of the track plots in the MWM test. O , P ELISA analysis of O Aβ40 and P Aβ42 levels in the plasma of mice. All the mice in the behavioural test and Aβ analysis were 12 months old and included WT (n = 6), Hmo-cys F + (n = 7), APP/PS1 (n = 7), and APP/PS1/Hmo-cys F + mice (n = 8). The data are presented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The Aβ40 and Aβ42 in human plasma were also determined by the ELISA kits (Elabscience Biotechnology Company, Wuhan, China, Cat No. E-EL-H0542; E-EL-H0543).

Techniques: Over Expression, Transgenic Assay, Control, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

doi: 10.1186/s12974-024-03119-2

Figure Lengend Snippet: Identification of the expression and structure of the cystatin F protein in the plasma of AD patients and analysis of its correlation with AD clinical manifestations. A Western blot analysis of the expression and structure of the cystatin F protein in plasma from patients with AD (n = 6) and controls (n = 6) by using a nondenaturing gel. The total protein in the plasma was stained with CBB as an internal control. B The structure of cystatin F in human plasma was determined by western blot assay, and DTT was added to the sample to reduce the dimers to the monomers. C ELISA analysis of secreted cystatin F levels in plasma from patients with AD (n = 45) and controls (n = 40). D ELISA analysis of secreted cystatin F in plasma according to the AD stage from mild (n = 15) to moderate (n = 15) to severe dementia (n = 15). E , F Correlations of the cystatin F level with the Aβ40 and Aβ42 levels in the plasma of patients with AD by Pearson correlation coefficient analysis. G -K Correlations of cystatin F levels with MMSE, MoCA, RAVLT-I, RAVLT-D and CDR scores by Pearson correlation coefficient analysis. The data are presented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The Aβ40 and Aβ42 in human plasma were also determined by the ELISA kits (Elabscience Biotechnology Company, Wuhan, China, Cat No. E-EL-H0542; E-EL-H0543).

Techniques: Expressing, Western Blot, Staining, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

doi: 10.1186/s12974-024-03119-2

Figure Lengend Snippet: Cystatin F dimers inhibited the uptake of Aβ by monocytes. A - C THP-1 cells were pretreated with cystatin F dimer protein for 30 min, and 1 μg/mL soluble Aβ42-Alexa Fluor 647, or Aβ42, or Aβ42-Alexa Fluor 555 were added for an additional 30 min. Then, the cells were subjected to A FCM, B ELISA and C LSCM analysis. D THP-1 cells were pretreated with 5 μg/mL cytochalasin D for 30 min, 40 μM EIPA for 1 h, and 250 ng/mL cystatin F for 30 min. Then, cells were incubated with 1 μg/mL soluble Aβ42-Alexa Fluor 647 for 30 min and analysed by FCM method. E THP-1 cells were pretreated with 250 ng/mL cystatin F, and 1 μg/mL soluble Aβ42-Alexa Fluor 555 was added, then cells were incubated for 30 min at 0 °C. Then, the cells were observed by LSCM. F , G Schematic diagram depicting the use of TIRFM for imaging membrane-localized proteins. THP-1 cells were pretreated with 250 ng/mL cystatin F dimer for 30 min, and then 1 μg/mL soluble Aβ42-Alexa Fluor 555 was added and incubated for 30 min at 0 °C. Cells were observed by TIRFM. H , I THP-1 cells were pretreated with 250 ng/mL cystatin F, and 1 μg/mL soluble Aβ42-Alexa Fluor 647 or Aβ42 was added, then cells were incubated for 30 min at 0 °C. Then, the cells were subjected to H FCM and I ELISA. The data are presented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 5 μm

Article Snippet: The Aβ40 and Aβ42 in human plasma were also determined by the ELISA kits (Elabscience Biotechnology Company, Wuhan, China, Cat No. E-EL-H0542; E-EL-H0543).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Imaging, Membrane

Journal: Journal of Neuroinflammation

Article Title: Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

doi: 10.1186/s12974-024-03119-2

Figure Lengend Snippet: Cystatin F dimers physically interact with Aβ to inhibit the internalization of Aβ by monocytes. A Schematic of the binding assay. B Plates were coated with 4 ng/μL dissolved Aβ40, Aβ42, Aβ40-1, and Aβ42-1 for 16 h. Recombinant cystatin F dimers were added to the wells for 3 h. The primary antibody for cystatin F and the secondary antibody coupled with HRP were used to generate a colour reaction, and the absorbance was measured at 450 nm. C , D The GST pull-down assay and His pull-down assay were used to analyse the interaction between the cystatin F dimer and Aβ. E Schematic diagram depicting the BiFC assay for detecting the interaction between the cystatin F dimer and Aβ. F The BiFC assay was carried out as described in the Materials and Methods section. Then, 200 ng/mL cystatin F dimer-VN and 200 ng/mL Aβ-VC protein were incubated at 37 °C for 2 h, and the fluorescence intensity was assessed at 529 nm. G Prediction of the amino acid interactions between cystatin F dimer and Aβ. H-J The potential amino acids on cystatin F that interacted with Aβ were mutated to alanine. The interaction was determined by a GST pull-down assay. K – L THP-1 cells were pretreated with 250 ng/mL cystatin F dimer protein or cystatin F (RTSTK) mutant protein for 30 min, 1 μg/mL Aβ42-Alexa Fluor 647 or Aβ42 was added for an additional 30 min, and the cells were subjected to K FCM and L ELISA analysis . M , N THP-1 cells were pretreated with 250 ng/mL cystatin F dimer protein or cystatin F (RTSTK) mutant protein for 30 min, and 1 μg/ml Aβ42-Alexa Fluor 555 or Aβ42-Alexa Fluor 647 were added for an additional 30 min, and the cells were observed by M TIRFM and subjected to N FCM . The data are presented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. ** p < 0.01, *** p < 0.001. Scale bar: 5 μm

Article Snippet: The Aβ40 and Aβ42 in human plasma were also determined by the ELISA kits (Elabscience Biotechnology Company, Wuhan, China, Cat No. E-EL-H0542; E-EL-H0543).

Techniques: Binding Assay, Recombinant, Pull Down Assay, Bimolecular Fluorescence Complementation Assay, Incubation, Fluorescence, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Monocytes release cystatin F dimer to associate with Aβ and aggravate amyloid pathology and cognitive deficits in Alzheimer’s disease

doi: 10.1186/s12974-024-03119-2

Figure Lengend Snippet: High-level cystatin F dimers in plasma rapidly aggravate cognitive impairment in 5xFAD transgenic mice. A Schematic of tail vein injection of 5xFAD mice, behavioural tests, and pathological analysis. B The purified murine cystatin F dimer was identified by western blot analysis. C The level of cystatin F in the plasma of the mice after tail vein injection of purified protein which was detected by ELISA analysis (n = 8). D Immunofluorescence and statistical analysis of Aβ plaques in brain section from transgenic mice that received a tail vein injection. Scale bar: 500 μm E , F ELISA method for measuring Aβ40 and Aβ42 levels in the brain of the mice. G , H ELISA method for measuring Aβ40 and Aβ42 levels in the plasma of the mice. I – L MWM analysis of the latency (s), number of target crosses, and mean speed (cm/s) in the invisible platform test of the mice. M Representative images of the track plots in the MWM test. N The motion traces of the NOR test were displayed. O The recognition index was calculated as described in Material and Methods. The data are presented as the means ± SD and were analysed using Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The Aβ40 and Aβ42 in human plasma were also determined by the ELISA kits (Elabscience Biotechnology Company, Wuhan, China, Cat No. E-EL-H0542; E-EL-H0543).

Techniques: Transgenic Assay, Injection, Purification, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Establishment of a sandwich ELISA for detecting L 59 LAP-DPs. a Schematic diagram of the ELISA for L 59 LAP-DPs. In the ELISA, L 59 LAP-DPs are first captured by coated L59 antibodies (Ab) and then sandwiched by biotin-conjugated anti-LAP antibodies (αLAP-Ab), which form a complex with strep-AP. For detection, an enzyme substrate is added and absorbance at 405 nm was measured. b , c Incubation time- and PLK concentration-dependent increases in the absorbance of the samples containing LAP incubated with PLK. The LAP (final 25 nM) was digested with 12.5 nM (open triangle), 25 nM (open reverse triangle), and 50 nM (open square) PLK, or without PLK (open circle) for 0–90 min, and then the samples were diluted by 1/50 and subjected to the L 59 LAP-DP ELISA ( b ). Also, 25 nM rhLAP was digested with 50 nM PLK (open square) or PLN (cross mark), or PLK in the presence of 5 μM camostat mesilate (open diamond) ( c ). All data were presented as mean ± SD from two different experiments ​*p-value <0.05, **p-value <0.01, ***p-value<0.001 obtained comparing to corresponding control values

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Correlation among L 59 LAP-DPs and active TGF-β in the culture medium, and intracellular signal transduction. a , c The ×9CAGA-Luc-transformed CCL64 cells were cultured in PLK-added CM derived from HEK293T cells overexpressing hLTGF-β1. After 6 h, the levels of active TGF-β1 and L 59 LAP-DPs were determined by respective ELISAs ( a ), and the extent of TGF-β signaling was measured by luciferase activity in CCL64 cells ( c ). b , d The scatterplots between the levels of L 59 LAP-DPs and active TGF-β shown in a ( b ) and between increases in L 59 LAP-DP levels and increases in luminescence from the values obtained compared to basal levels in the absence of PLK shown in c ( d ). A significant positive correlation was seen ( b ) * p-value <0.05, ***p-value <0.001 obtained comparing to 0

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Transformation Assay, Cell Culture, Derivative Assay, Luciferase, Activity Assay