Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: Nrf2 suppresses TLR4 and IRF1 levels and promotes M2Mϕ polarization in vitro . (A) BMDM and COM-TECs coculture schematic diagram. Western blot (B) and qPCR (C) analyses of Nrf2, TLR4, IRF1, iNOS, and ARG-1 levels in BMDMs co-cultured with increasing COM dose stimulated TECs. GAPDH was used as an internal control. (D, F) Western blot detection of Nrf2, TLR4, IRF1, iNOS, and ARG-1 levels following SFN treatment or Nrf2 upregulation/downregulation in BMDMs co-cultured with COM-stimulated TECs. GAPDH was used as an internal control. (E, G) The distribution of iNOS (green) and ARG-1 (red) in BMDMs according to immunofluorescence. (H, I) Flow cytometric analysis of the polarization state of BMDMs using anti-CD11c and anti-CD206 in F4/80 + and CD11b + cells. The data are shown as the mean ± standard error (SE) of three independent experiments. *P < 0.05; **P < 0.01, as determined by Student's t test (C) or one-way ANOVA (E, G, H, I).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: In Vitro, Western Blot, Cell Culture, Control, Immunofluorescence

Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: Nrf2 inhibits TLR4 and IRF1 expression by directly binding to miR-93 promoter. (A) miRNA-array heatmap revealed the top 30 LPS-regulated miRNAs in BMDMs. Additionally, miRNAs predicted to be transcriptionally activated by Nrf2 (based on the JASPRA database) are highlighted. (B) Venn analysis identified miRNAs that could target TLR4 and IRF1 and be transcriptionally activated by Nrf2. (C) Renal expression of mmu-miR-93-5p following treatment with an Nrf2-neutralizing antibody or SFN in mice with CaOx nephrocalcinosis was detected using FISH (200× magnification for all panels; scale bar: 20 µm). (D) qPCR analysis of the expression levels of mmu-miR-93-5p in BMDMs. U6 RNA was detected as an internal control. (E, F) ChIP assays and ChIP qPCR analysis of Nrf2 binding to the predicted miR-93 ARE in BMDMs treated with Nrf2. (G) A schematic model showing Nrf2 directly binds the promoter of miR-93 and activates its transcription. (H, I) Schematic representation of mutant and WT seed sequences of miR-93 targeting the 3' UTRs of TLR4 and IRF1. Luciferase reporters harboring putative target sites in WT and mutant 3' UTRs of TLR4 (J) or IRF1 (L) were co-transfected with miR-93 mimics (100 nM). TLR4 (K) and IRF1 (M) expression detected by qPCR in BMDMs transfected with miR-93 mimics or inhibitor. Western blot (N) and qPCR (O) detection of TLR4 and IRF1 levels, as well as the Mϕ-polarization markers iNOS and ARG-1 in BMDMs transfected with miR-93 mimics or inhibitor. GAPDH was used as an internal control. Data represent the mean ± standard error (SE) of three independent experiments. *P < 0.05; **P < 0.01, as determined by Student's t test (D, F) or one-way ANOVA (J-M, O).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: Expressing, Binding Assay, Control, ChIP-qPCR, Mutagenesis, Luciferase, Transfection, Western Blot

Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: SFN dependeds on the Nrf2-miR-93-TLR4/IRF1 axis to suppress TLR4 and IRF1 expression and promote M2Mϕ polarization in vitro . Western blot (A) and qPCR (B) analyses of Nrf2, TLR4, IRF1, iNOS, and ARG-1 levels in BMDMs treated with SFN and/or miR-93 inhibitor. GAPDH was used as an internal control. (C) The distribution of iNOS (green) and ARG-1 (red) in BMDMs treated with SFN and/or miR-93 inhibitor according to immunofluorescence (200× magnification; scale bar: 20 µm). (D) Fluorescence microscopy analysis of BMDM phagocytic ability. COM crystals were labeled with an Alexa Fluor 488-conjugated IgG and directly cultured with treated BMDMs (200× magnification; scale bar: 20 µm). Flow cytometric analysis of BMDM polarization (E) and TECs necrosis (F) in co-cultured cells treated with SFN and/or miR-93 inhibitor. Data represent the mean ± standard error (SE) of three independent experiments. *P < 0.05; **P < 0.01, as determined by one-way ANOVA (B-G).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: Expressing, In Vitro, Western Blot, Control, Immunofluorescence, Fluorescence, Microscopy, Labeling, Cell Culture

Journal: Theranostics

Article Title: Sulforaphane elicts dual therapeutic effects on Renal Inflammatory Injury and crystal deposition in Calcium Oxalate Nephrocalcinosis

doi: 10.7150/thno.44054

Figure Lengend Snippet: SFN dependeds on the Nrf2-miR-93-TLR4/IRF1 axis to suppress CaOx crystal deposition and kidney injury in vivo . (A) Diagram of the experimental design. (B) Polarized-light optical microscopy detection of renal CaOx crystal deposition in mice treated with SFN and/or antagomiR-93 (20× magnification; scale bar: 500 µm). Pizzolato staining to detect corticomedullary CaOx crystal deposition. PAS and TUNEL staining to detect renal tubular epithelial cell injury (200× magnification; scale bar: 20 µm). (C) 18 F-FDG PET-CT scanning to detect kidney inflammation in CaOx nephrocalcinosis mice treated with SFN and/or antagomiR-93. (D) IHC detection of Nrf2, TLR4, and IRF1 levels (400× magnification; scale bar: 40 µm) and FISH detection of miR-93 expression in kidney tissue (200× magnification; scale bar: 20 µm). (E) The distributions of iNOS (red) and ARG-1 (green) in kidney tissues according to immunofluorescence. (F) Serum levels of the proinflammatory cytokines IL-1β, TNF-α, and IL-6 and the anti-inflammatory cytokine IL-10 according to ELISA on days 3, 4, and 10. *P < 0.05; **P < 0.01, as determined by one-way ANOVA (B-F).

Article Snippet: Protein extract was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated overnight at 4°C with primary antibodies against Nrf2 (AF7006; 72 kDa; 1:1000; Affinity Biologicals), TLR4 (GB11519; 95 kDa; 1:1000; Servicebio), IRF1 (abs118047; 37 kDa; 1:1000; Absin), iNOS (BA0362; 130 kDa; 1:200; Boster), arginase 1 (ARG-1; GB11285; 35-40 kDa; 1:5000; Servicebio), or GAPDH (T0004; 34 kDa; 1:5000; Affinity Biologicals).

Techniques: In Vivo, Microscopy, Staining, TUNEL Assay, Positron Emission Tomography-Computed Tomography, Expressing, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Identification and Validation of miRNA-TF-mRNA Regulatory Networks in Uterine Fibroids

doi: 10.3389/fbioe.2022.856745

Figure Lengend Snippet: The proliferation of UF cells regulated by (A) overexpression or (B) slience of hub DEMs was detected by CCK-8 assay. (C) The migrational ability of UF cells regulated by overexpression or slience of hub DEMs was detected by Transwell. (D) The apoptosis rates of UF cells regulated by overexpression or slience of hub DEMs was detected by AV/PI assay.

Article Snippet: To determine the effect of hub DEMs on the apoptosis of leiomyoma cells, cells were harvested and centrifuged for annexin V-FITC/PI apoptosis detection (Boster Biotech Co., Ltd., Wuhan, China) after 24 h of transfection.

Techniques: Over Expression, CCK-8 Assay

Journal: Oncoimmunology

Article Title: Oncolytic measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by human myeloid and plasmacytoid dendritic cells

doi: 10.1080/2162402X.2016.1261240

Figure Lengend Snippet: MV induces TRAIL expression and IFN-α secretion by pDCs and CD1c + DCs. pDCs were cultured with IL3, IL3+MV or the TLR7 agonist R837 (A). CD1c + DCs were cultured alone (−), with MV or R837 (B). The expression of surface markers by the indicated cells was determined by flow cytometry. IFN-α secretion was measured by ELISA. #, values are below the limit of detection of the kit (7 pg/mL). Results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, one-way ANOVA (Kruskal–Wallis).

Article Snippet: DCs were also cultivated with the TLR7 agonist R837 (1 μg/mL, Invivogen), exogenous type I IFNs (rhIFN-α-2a and rhIFN-β-1a, 100 ng/mL, ImmunoTools) or the RIG-I agonist 5′-ppp-dsRNA LyoVec (10 μg/mL, Invivogen).

Techniques: Expressing, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Oncoimmunology

Article Title: Oncolytic measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by human myeloid and plasmacytoid dendritic cells

doi: 10.1080/2162402X.2016.1261240

Figure Lengend Snippet: TRAIL expression depends on IFN-α secreted by DCs exposed to MV. pDCs (A) and CD1c + DCs (B) were pretreated or not with Ruxolitinib (Rux) before exposure to IL3+MV or MV respectively, or before exposure to R837. The secretion of IFN-α was measured by ELISA. #, values are below the limit of detection of the kit (7 pg/mL). The expression of TRAIL by the indicated cells was determined by flow cytometry. (C) pDCs and CD1c + DCs were pretreated or not with Rux before exposure to type I IFNs (rhIFN-α-2a and rhIFN-β-1a). TRAIL positive cells were quantified by flow cytometry. Results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, Mann–Whitney test.

Article Snippet: DCs were also cultivated with the TLR7 agonist R837 (1 μg/mL, Invivogen), exogenous type I IFNs (rhIFN-α-2a and rhIFN-β-1a, 100 ng/mL, ImmunoTools) or the RIG-I agonist 5′-ppp-dsRNA LyoVec (10 μg/mL, Invivogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Oncolytic measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by human myeloid and plasmacytoid dendritic cells

doi: 10.1080/2162402X.2016.1261240

Figure Lengend Snippet: MV induces IFN-α and TRAIL expression following RLR activation in CD1c + DCs and RLR/TLR7 activation in pDCs. (A) pDCs were pretreated or not with IRS661 (TLR7 inhibitor) or with MRT67307 (TBK1 and IKK-ε inhibitor) and then cultured with IL3, IL3+MV or R837. (B) CD1c + DCs were pretreated or not with IRS661 or MRT67307 and then cultured alone (−) or with MV. (C) pDCs were cultured with IL3+MV or with IL3+UV-inactivated MV (MV UV*) and CD1c + DCs were cultured alone (−), with MV or MV UV*. (D) pDCs and CD1c + DCs were exposed to 5′-ppp-dsRNA LyoVec, a RIG-I agonist. (A–D) IFN-α secretion was measured by ELISA. #, values are below the limit of detection of the kit (7 pg/mL). The expression of TRAIL by the indicated cells was determined by flow cytometry. Results are expressed as the mean ± SEM of three independent experiments. (A, B) * p < 0.05, one-way ANOVA (Kruskal–Wallis). (C) * p < 0.05, Mann–Whitney test.

Article Snippet: DCs were also cultivated with the TLR7 agonist R837 (1 μg/mL, Invivogen), exogenous type I IFNs (rhIFN-α-2a and rhIFN-β-1a, 100 ng/mL, ImmunoTools) or the RIG-I agonist 5′-ppp-dsRNA LyoVec (10 μg/mL, Invivogen).

Techniques: Expressing, Activation Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Oncolytic measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by human myeloid and plasmacytoid dendritic cells

doi: 10.1080/2162402X.2016.1261240

Figure Lengend Snippet: MV induces IFN-α and TRAIL expression after RIG-I activation in Gen2.2 cells. Gen2.2 cells established by transduction with lentiviruses expressing scrambled shRNA, shRNA against TLR7 (shTLR7) or RIG-I (shRIG-I) were cultured with IL3, IL3+MV or IL3+UV-inactivated MV (MV UV*). IFN-α secretion was measured by ELISA. #, values are below the limit of detection of the kit (7 pg/mL).The percentage of TRAIL positive Gen2.2 cells was determined by flow cytometry. Results are expressed as the mean ± SEM of three independent experiments. * p < 0.05, one-way ANOVA (Kruskal–Wallis).

Article Snippet: DCs were also cultivated with the TLR7 agonist R837 (1 μg/mL, Invivogen), exogenous type I IFNs (rhIFN-α-2a and rhIFN-β-1a, 100 ng/mL, ImmunoTools) or the RIG-I agonist 5′-ppp-dsRNA LyoVec (10 μg/mL, Invivogen).

Techniques: Expressing, Activation Assay, Transduction, shRNA, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Oncoimmunology

Article Title: Oncolytic measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity by human myeloid and plasmacytoid dendritic cells

doi: 10.1080/2162402X.2016.1261240

Figure Lengend Snippet: DCs activated with MV acquire a functional cytotoxic activity. (A) pDCs were pretreated or not with IRS661 or Rux and then cultured with IL3+MV, R837, type I IFNs (rhIFN-α-2a and rhIFN-β-1a) or 5′-ppp-dsRNA LyoVec. (B) CD1c + DCs were pretreated or not with IRS661 or Rux and then cultured with MV, UV-inactivated MV, R837, type I IFNs (rhIFN-α-2a and rhIFN-β-1a) or 5′-ppp-dsRNA LyoVec. (A, B) DCs were then added to the target Jurkat cells at an effector:target ratio of 20:1 and the percentage of specific lysis of Jurkat cells was determined by the measure of 51 Cr release in the supernatants. Results are expressed as mean ± SEM of at least three independent experiments and each condition was assessed in triplicates for each experiment. For the left panel: * p < 0.05, one-way ANOVA (Kruskal–Wallis); for the right panel: * p < 0.05, Mann–Whitney test.

Article Snippet: DCs were also cultivated with the TLR7 agonist R837 (1 μg/mL, Invivogen), exogenous type I IFNs (rhIFN-α-2a and rhIFN-β-1a, 100 ng/mL, ImmunoTools) or the RIG-I agonist 5′-ppp-dsRNA LyoVec (10 μg/mL, Invivogen).

Techniques: Functional Assay, Activity Assay, Cell Culture, Lysis, MANN-WHITNEY

Journal: The Journal of comparative neurology

Article Title: The distribution, number and certain neurochemical identities of infracortical white matter neurons in a lar gibbon ( Hylobates lar ) brain.

doi: 10.1002/cne.24545

Figure Lengend Snippet: Sources and dilution of antibodies used in the current study.

Article Snippet: nNOS , rabbit , Recombinant human neuronal nitric oxide synthase , Merck-Millipore , AB5380 , Russo et al., 2013 , 1:6000 , AB_91824.

Techniques: Recombinant

Journal: The Journal of comparative neurology

Article Title: The distribution, number and certain neurochemical identities of infracortical white matter neurons in a lar gibbon ( Hylobates lar ) brain.

doi: 10.1002/cne.24545

Figure Lengend Snippet: Stereological parameters used for estimating neuronal numbers in the white matter of the lar gibbon. NeuN – neuronal nuclear marker; nNos – neuronal nitric oxide synthase; CR – calretinin.

Article Snippet: nNOS , rabbit , Recombinant human neuronal nitric oxide synthase , Merck-Millipore , AB5380 , Russo et al., 2013 , 1:6000 , AB_91824.

Techniques: Marker, Sampling

Journal: The Journal of comparative neurology

Article Title: The distribution, number and certain neurochemical identities of infracortical white matter neurons in a lar gibbon ( Hylobates lar ) brain.

doi: 10.1002/cne.24545

Figure Lengend Snippet: Frequency distribution bar plots of somal volumes of WMICs in the brain of the lar gibbon. (a) Volumes of the soma of WMICs immunoreactive to neuronal nuclear marker (NeuN). Note the median volume is 615.9 μm3, with a range from 63.6 to 3 716.6 μm3. (b) Volumes of the soma of WMICs immunoreactive to neuronal nitric oxide synthase (nNOS). Note the median volume is 817.8 μm3, with a range from 110.6 to 2 899.8 μm3. (c) Volumes of the soma of WMICs immunoreactive to calretinin. Note the median volume is 619.4 μm3, with a range from 56.2 to 1 952.7 μm3. In all plots the bin width = 92.9 μm3.

Article Snippet: nNOS , rabbit , Recombinant human neuronal nitric oxide synthase , Merck-Millipore , AB5380 , Russo et al., 2013 , 1:6000 , AB_91824.

Techniques: Marker

Journal: The Journal of comparative neurology

Article Title: The distribution, number and certain neurochemical identities of infracortical white matter neurons in a lar gibbon ( Hylobates lar ) brain.

doi: 10.1002/cne.24545

Figure Lengend Snippet: Photomicrographs of neuronal nitric oxide synthase (nNOS) immunostaining in the rostral portion of the frontal lobe of the lar gibbon showing the distribution of WMICs immunoreactive to nNOS. (a) Moderately magnified image of the superior frontal gyrus (from the region indicated by the b in Fig. 1a), showing the numerous nNOS-immunoreactive WMICs deep to the cerebral cortex (grey matter, GM, white matter, WM). The approximate boundary of the deep border of cortical layer VI and the WM is marked by a dashed line. (b) High magnification image of the cortical/white matter boundary (marked by a dashed line) of the superior frontal gyrus, showing the nNOS-immunoreactive WMICs deep to the cerebral cortex. Arrows in a and b indicate the same neuron for orientation of image location. (c) Moderately magnified image of the fundus of the inferior frontal gyrus (from the region indicated by the d in Fig. 1a), showing the nNOS-immunoreactive WMICs deep to the cerebral cortex (GM) within the WM. The approximate boundary of the deep border of cortical layer VI and the WM is marked by a dashed line. (d) High magnification image of the cortical/white matter boundary (marked by a dashed line) of the fundus of the inferior frontal gyrus, showing the nNOS-immunoreactive WMICs deep to the cerebral cortex within the WM. Arrows in c and d indicate the same neuron for orientation of image location. Scale bar in c = 500 μm and apply to a and c. Scale bar in d = 250 μm and applies to b and d. In all images dorsal is to the top of the image and medial to the left.

Article Snippet: nNOS , rabbit , Recombinant human neuronal nitric oxide synthase , Merck-Millipore , AB5380 , Russo et al., 2013 , 1:6000 , AB_91824.

Techniques: Immunostaining