Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 4 The effect of monosodium urate (MSU) crystals on Caspase 1 activity and IL-1β secretion is time-of-day dependent. (A) Schematic of the experimental design to assess the time-of-day differences in NLRP3 inflammasome activity. Following 18 h serum starvation, cells were media changed to serum-replete media and either treated immediately with MSU crystals (500 μg/mL) or rested for 12 h before media-changing again with/without MSU crystal addition. Caspase 1 activity was measured following 12 h of MSU crystal exposure and normalized for differences in cell number as determined by CyQuant assay with reference to a standard curve. (B) The magnitude of Caspase 1 activity induced by MSU crystal exposure in cells at the 0–12 h compared with 12–24 h timepoints. (C) The magnitude of increase in secreted IL-1β levels induced by MSU crystal exposure in THP-1 macrophages at the 0–12 h compared with 12–24 h timepoints. Data shown are mean ± SEM. Data were analyzed by t-test with p < .05 considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Activity Assay, CyQUANT Assay

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 6 BMAL1 represses expression of pro-IL-1β and CASP1 but not NLRP3 in THP-1 macrophages BMAL1 was knocked down or over-expressed in THP-1 macrophages using adenoviral-mediated gene delivery. Following gene transduction, cells were serum starved for 18 h then media changed to serum-replete media and either treated immediately with MSU crystals (500 μg/mL) (0–12 h timepoint) or rested for 12 h before media-changing again with/without MSU crystal addition (12–24 h timepoint). NLRP3 expression measured by RT-qPCR following BMAL1 knockdown at (A) the 0–12 h timepoint and (B) 12–24 h timepoint. NLRP3 expression measured by RT-qPCR following BMAL1 overexpression at (C) the 0–12 h timepoint and (D) 12–24 h timepoint. Pro-IL-1β expression measured by RT-qPCR following BMAL1 knockdown at (E) the 0–12 h timepoint and (F) 12–24 h timepoint. Pro-IL-1β expression measured by RT-qPCR following BMAL1 overexpression at (G) the 0–12 h timepoint and (H) 12–24 h timepoint. CASP1 expression measured by RT-qPCR following BMAL1 knockdown at (I) the 0–12 h timepoint and (J) 12–24 h timepoint. CASP1 expression measured by RT-qPCR following BMAL1 overexpression at (K) the 0–12 h timepoint and (L) 12–24 h timepoint. Data shown are mean ± SEM for 3 experimental replicates. Data were analyzed by one-way ANOVA (post-hoc Tukey) with p < .05 considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Knockdown, Over Expression

Journal: The FASEB Journal

Article Title: Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare

doi: 10.1096/fj.202202035r

Figure Lengend Snippet: FIGURE 7 REV-ERBα represses NLRP3 expression and inflammasome activity in THP-1 macrophages. Following 18 h serum starvation, cells were re-fed with serum-replete media and either treated immediately with/without MSU crystals (500 μg/mL) and heme (30 μM)), SR8278 (1 μM), heme+SR8278 or vehicle (0.1M NaOH + 0.1% DMSO) for 12 h (0–12 h timepoint) or rested for 12 h and then media-changed to fresh serum-replete media with/without MSU crystals and heme/SR8278/vehicle (12–24 h timepoint). (A) Caspase-1 activity normalized to cell number as measured at the 0–12 h timepoint and (B) 12–24 h timepoint. (C) Levels of secreted IL-1β measured by ELISA at the 0–12 h and (D) 12–24 h timepoint. To ensure all cells were exposed to the same vehicles, SR8278 vehicle (0.1%) DMSO) was added to the heme-only treatment and heme vehicle (0.1M NaOH) added to the SR8278-only treatment. Data shown are mean ± SEM for 3 experimental replicates. All data were analyzed by one-way ANOVA (post-hoc Tukey) p < .05 was considered statistically significant.

Article Snippet: Secreted IL- 1β protein levels were measured in the cell culture supernatant using a Duoset Human IL- 1β ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's protocol.

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of biomedical materials research. Part A

Article Title: In vitro evaluation of bone cell response to novel 3D-printable nanocomposite biomaterials for bone reconstruction.

doi: 10.1002/jbm.a.37719

Figure Lengend Snippet: FIGURE 5 Differentiated MC3T3-E1 osteoblast cell proliferation on mSLA-printed nanocomposite discs. SP10 (10% nanohydroxyapatite [nHA]), mSP10 (methacrylated acrylated epoxidized soybean oil [mAESO] and 10% nHA), SP30 (30% nHA), mSP30 (mAESO and 30% nHA), and hydroxyapatite (HA) (control). Proliferation of dMC3T3-OB cells as measured by XTT assay. Data are means ± SD (n = 8 for nanocomposite discs and n = 5 for HA discs, from four experiments), (statistically significant, *p ≤.05, **p ≤.01, ***p ≤.001).

Article Snippet: Cell proliferation after 1, 3, and 7 days of culture was characterized using XTT (sodium 30-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy6-nitro) benzene sulfonic acid hydrate; TACS XTT cell proliferation kit, R&D Systems, Minneapolis, USA).

Techniques: Control, XTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Thrombospondin 1–CD47 Signalling Modulates Vascular Smooth Muscle Cell Senescence in Chronic Kidney Disease

doi: 10.3390/ijms27020755

Figure Lengend Snippet: TSP1 and indoxyl sulfate limit VSMC proliferation via CD47. hVSMC cell viability was measured by assessing reduction in tetrazolium salt sodium 3′- [1- [(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) and measuring absorbance at 450 nm in cells after 48 h treatment with ( A ) TSP1 (0, 0.2, 2.2, 5, 10 nM) ( n = 4), ( B ) TSP1 2.2 nM ± pre-treatment with anti-CD47 antibody for 30 min ( n = 6), ( C ) IS (0, 1, 10, 100, 500 µM) ( n = 4), or ( D ) IS (100, 500 µM) ± pre-treatment with anti-CD47 antibody for 30 min ( n = 4–8). All data shown are mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 by one-way analysis of variance with Holm–Sidak post-hoc test ( A , C ) or Kruskal–Wallis test ( B , D ). Abbreviations: αCD47—anti-CD47 antibody; hVSMC—human aortic vascular smooth muscle cell; IS—indoxyl sulfate; TSP1—thrombospondin-1.

Article Snippet: Cell proliferation and senescence were measured using XTT Cell Viability Kit (#9095, Cell Signalling Technology, Danvers, MA, USA) and Mammalian β-Galactosidase Assay Kit (75707, Thermo Fisher Scientific), respectively.

Techniques:

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-primed ADSCs (vADSCs) promoted the viability, anchorage independent growth, migration, invasion, epithelial-mesenchymal transition (EMT), and stemness property of breast cancer cells. ADSCs were treated with or without visfatin, noted as vADSCs or uADSCs, respectively, at 50 and 100 ng/mL for 48 h. Then, MDA-MB-231 cells were indirectly co-cultured with uADSCs or vADSCs in a transwell system for 72 h, noted as Ctrl or V50 and V100. ( A ) After that, the MDA-MB-231 collected from the co-culture were seeded in 96-well plate for 24, 48, and 72 h for analyzing the cell viability by XTT assay. ( B ) The collected MDA-MB-231 cells were seeded in a six-well plate with Noble agar for 21 days to analyze the anchorage independent growth by soft agar colony formation assay. ( C ) The collected MDA-MB-231 cells were seeded in a 24-well transwell plate coated with or without the Matrigel for performing migration or invasion assay, respectively. ( D ) The collected MDA-MB-231 cells were seeded in a 96-well low binding plate for tumorsphere formation. ( E ) The collected MDA-MB-231 cells were analyzed for the expressions of EMT- and stemness-related proteins by western blotting. MDA-MB-231 cells only were noted as Alone. Representative images of western blot shown. The result was quantified and present as histogram. All experiments were performed in triplicate. The statistical differences were calculated by t-test from three independent experiments. p -values < 0.05 or < 0.01 were marked with “*” or “**”, respectively.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Migration, Cell Culture, Co-Culture Assay, XTT Assay, Soft Agar Assay, Invasion Assay, Binding Assay, Western Blot

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: GDF15 play a crucial role in the regulations of breast cancer cells activities by visfatin-treated ADSCs (vADSCs). ( A ) After the three-day co-culture of MDA-MB-231 cells and vADSCs (V50 and V100 group) or untreated ADSCs (Ctrl group), the CM was collected and analyzed by using a cytokine array kit. ( B ) The expression of GDF15 in the co-cultured CM was validated by ELISA represented in a histogram. The ADSCs and MDA-MB-231 cells collected from the co-culture system were extracted for cell lysate to analyze the GDF15 expression by western blotting. ( C ) The migration and invasion of MDA-MB-231 treated with GDF15 at various concentrations for 48 h were evaluated by using a transwell system. ( D ) The indirect co-culture was performed in the presence or absence of the GDF15 neutralizing antibody of for three days. After that, the migration and invasion of the MDA-MB-231 cells collected from the co-culture were evaluated in a transwell system. ( E ) The expression of phosphor-AKT (pAKT) of MDA-MB-231 treated with GDF15 (50 ng/mL) at different time point was detected by western blotting. ( F ) The pAKT was detected in the MDA-MB-231 cells from the co-culture by western blotting. ( G ) After the three-day co-culture in the presence or absence of the wortmannin (400 nM), the MDA-MB-231 cells were collected from the co-culture for performing the migration assay. All experiments were performed in triplicate.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Co-Culture Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-primed ADSCs promoted the tube formation of HUVEC. ( A ) HUVEC cells were co-cultured with visfatin-treated ADSCs or untreated ADSCs, noted as V50 and V100 or Ctrl, respectively, for three days. The HUVEC cells were collected from the co-culture and seeded in a matrix gel-coated 96-well plate. The tube formation of HUVEC was observed using a microscope. The length of branches was determined by using the ImageJ software. ( B ) After the three-day co-culture in the presence or absence of GDF15 neutralizing antibody (GDF15 Nab, 5 μg/mL), the HUVEC cells were collected from the co-culture for performing the tube formation assay. The experiments were performed in triplicate.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Cell Culture, Co-Culture Assay, Microscopy, Software, Tube Formation Assay

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-pretreated ADSCs (vADSCs) enhanced the tumor growth and metastasis in human breast cancer xenograft mouse model. ( A ) The nude mice were injected with mixture of MDA-MB-231 and untreated ADSCs (uADSCs) or vADSCs, noted as Ctrl or V50, respectively, to the mammary fat pads. The tumor volumes were measured every week after injection (Ctrl, n = 5; V50, n = 6). ( B ) After sacrificing the mice, the weight of the resected tumor was measured. ( C ) The expressions of GDF15, β-catenin, CD31, and pAKT in the tumor sections were detected by immunohistochemistry. The IHC score was calculated by multiplying the percentage of positive cells by the intensity and present as histogram. ( D ) The luciferase-expressing MDA-MB-231 were collected and injected into the tail vein of NOD/SCID mice after co-culturing with uADSCs or vADSCs, noted as Ctrl or V50, respectively (Ctrl, n = 8; V50, n = 8). The IVIS radiance signals of the mice were assessed at week 4. The representative images of high and low signal were shown. The statistical differences were calculated by t-test, *, p -value < 0.05; **, p -value < 0.01.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Injection, Immunohistochemistry, Luciferase, Expressing

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: The expressions of visfatin, GDF15, and pAKT in the specimens from breast cancer patients. ( A ) The expressions of visfatin, GDF15, and pAKT in breast cancer tissue microarray (n = 96) were detected by immunohistochemistry. The representative images of high expression levels (No. 1) and low expression levels (No. 2) were shown. The IHC score was calculated by multiplying the percentage of positive cells by the intensity, which was identified using HistoQuest Analysis Software. ( B ) The correlations between visfatin, GDF15, and pAKT according to the IHC score were calculated by using the online Pearson correlation coefficient calculator. ( C ) The correlation of serum levels of GDF15 and visfatin of breast cancer patients (n = 120) determined by ELISA was also calculated by using the online Pearson correlation coefficient calculator.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Microarray, Immunohistochemistry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin mediates its effects both directly via cAbl/STAT3 and indirectly mediated by ADSCs via GDF15/AKT on promoting malignant behavior in breast cancer. Previously, we discovered visfatin mainly produced by adipocytes promoted breast cancer cells directly through activation of c-Abl and STAT3, which was blocked by Imatinib and Stattic inhibitor, respectively (black arrow). In this study, we showed that visfatin can act via an indirect pathway by priming ADSCs, which may be recruited from the adipose tissue to tumor site or generated from autologous fat transfer, to produce GDF15 that stimulated AKT activation in breast cancer cells to promote malignant behaviors (white arrow). The effect can be blocked by the treatment of GDF15 neutralizing Ab or Wortmannin inhibitor.

Article Snippet: The visfatin expression in the serum of breast cancer patients were detected by using a human visfatin ELISA kit (DY4335-05, R&D Systems).

Techniques: Produced, Activation Assay, Generated