Journal: Journal of proteome research

Article Title: Proteomic analysis reveals novel mechanisms by which polychlorinated biphenyls compromise the liver promoting diet-induced steatohepatitis

doi: 10.1021/acs.jproteome.8b00886

Figure Lengend Snippet: A. (i) Immunoblot analysis, and (ii) RT qPCR analysis of hepatic HNF4α protein and mRNA from mice fed a control diet (CD), control diet and exposed to Aroclor 1260 (20 mg/kg) (CD+), fed a HFD (HFD), and fed a HFD and exposed to Aroclor 1260 (20 mg/kg) (HFD+). B. RT qPCR Analysis of HNF4α target genes (i) Albumin, (ii) Cyp2c29, and (iii) Ttpa from murine liver. C. Western blot analysis of (i) Y1173 EGFR and (ii) HNF4α S313 phosphorylation in AML-12 cell lysates exposed to DMSO (0.1%), EGF (1.2 nM), and EGF+A1260 (1.2 nM EGF, 10 μg/mL). D. RT qPCR analysis of HNF4α target gene Pklr in AML-12s after 6-hour incubation with either DMSO (0.1%), EGF (1.2 nM), EGF+A1260 (1.2nM EGF, 10 μg/mL), or A1260 (10 μg/mL). All data are represented as box and whisker plots. An n=5 was used for the HNF4α protein levels analysis in vivo, an n=10 for Hnf4α mRNA and HNF4α target gene mRNA in vivo, an n=4 for HNF4α S313 phosphorylation, and an n=4 for Pklr mRNA. A P<0.05 is denoted with *. In the in vivo datasets an a denotes significance due to Aroclor, b HFD, and c interaction. Two-way ANOVA was used to statistically compare the in vivo data. One-way ANOVA was used for the statistical analysis for Fig 3Ci-ii. A two-way ANOVA was used for the statistical analysis in Fig 3D; an a denotes significance due to EGF, b due to Aroclor 1260.

Article Snippet: Hepatocyte nuclear factor 4 alpha (HNF4α) phosphorylation assay AML-12 (CRL-2254) cells obtained from ATCC (Manassas, VA) were exposed to DMEM/F-12 media containing either EGF (1.2 nM) (EMD Millipore, Burlingtion, MA), Aroclor 1260 (10 μg/mL) and EGF (1.2 nM), or 0.1% DMSO for 30 minutes then protein was extracted to measure phosphorylated HNF4α, total HNF4α, phosphorylated EGFR and total EGFR.

Techniques: Western Blot, Quantitative RT-PCR, Control, Phospho-proteomics, Incubation, Whisker Assay, In Vivo

Journal: Inflammation

Article Title: NOX2-Dependent Phagocyte Signaling Mediates Silica-Induced Murine Lung Injury via Macrophage-Neutrophil Interactions

doi: 10.1007/s10753-025-02389-z

Figure Lengend Snippet: NIP inhibited the release of NETs, but not cell death, in cultured neutrophils with SiNPs exposure. ( A ) Live cell imaging analysis of neutrophils isolated from bone marrow of mice that had been exposed to SiNPs in the presence or absence of NIP. Six hours after exposure, cells were stained with SYTOX green and observed by fluorescence microscopy. Scale bars at low magnification (top panels) = 100 μm and at high magnification (bottom panels) = 20 μm. ( B ) Neutrophils with antibodies to NE were stained with SYTOX green. Yellow arrowheads indicate extracellular signals of NE and SYTOX green, suggesting that neutrophils releasing NETs were significantly suppressed in the SiNPs + NIP group. Scale bars = 10 μm. ( C ) Neutrophils were immunostained with antibodies to PAD4 with Hoechst33342. Scale bars = 20 μm. D and E. Relative quantification of immunostaining experiments was depicted in violin plots. ( D ) NE signals NE signals relative to SYTOX signals. ( E ) PAD4 signals relative to nuclei Hoechst signals. For Figures, D and E, * p < 0.05, *** p < 0.001, and **** p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test

Article Snippet: Lung sections were reacted with anti-Gr-1 antibody (ab25377, 1:200; Abcam, Cambridge, UK) overnight, and reacted with anti-rat biotin-conjugated IgG antibody (BA-9400; Vector Laboratories, Newark, CA, USA) at room temperature for 1 h. And then, the slides were washed with PBS, reacted with an Alexa Fluor 594-conjugated streptavidin antibody (S32356, 1:200; Invitrogen, Carlsbad, CA, USA) at room temperature for 1 h. After nuclear staining with Hoechst 33,342 (346–07951, Dojindo, Kumamoto, Japan), the slides were mounted and photographed using BZ-9000 microscope.

Techniques: Cell Culture, Live Cell Imaging, Isolation, Staining, Fluorescence, Microscopy, Quantitative Proteomics, Immunostaining, Comparison

Journal: bioRxiv

Article Title: Multiview super-resolution microscopy

doi: 10.1101/2021.05.21.445200

Figure Lengend Snippet: a) Triple-view reconstruction of whole fixed adult C. elegans , labeled with NucSpot Live 488. Axial maximum intensity projection is shown. b-e) Comparative higher magnification views of dashed yellow rectangular region in a) , with bottom deconvolved view b) , commercial Leica SP8 confocal microscope c) , conventional triple-view deconvolution d) , attenuation-compensated triple-view deconvolution e) . Colored arrows highlight comparisons, orange: single-vs. triple-view, magenta: deconvolution methods. f) Triple-view reconstruction (green) with segmented nuclei overlaid in red (red), corresponding to red dashed rectangular region in a) . See also Movie S2. g) Schematic of larval wing disc, lateral (top) and axial (bottom) views, including adult muscle precursor myoblasts and notum. h) Lateral plane from triple-view reconstruction, 30 μm from sample surface. Notum nuclei (NLS-mCherry, magenta) and myoblast membranes (CD2-GFP, cyan) labeled. i) Axial maximum intensity projection derived from 6 μm thick yellow rectangle in h). j, k) Higher magnification view of white dashed line/rectangle in h/i) , comparing triple-view result j) to single View C deconvolution k) . White arrows: membrane observed in j) but absent in k). l) Schematic of kidney: approximate region where tissue was extracted. m) Four color triple-view reconstruction of mouse kidney slice. Lateral image at indicated axial height from beginning of the volume, highlighting glomerulus surrounded by convoluted tubules. Red: nuclei stained with DAPI, green: actin stained with phalloidin-Alexa Fluor 488; magenta: tubulin immunolabeled with mouse-α-Tubulin primary, α-Mouse-JF549 secondary; yellow: CD31 immunolabeled with Goat-α-CD31 primary, α-Goat AF647 secondary. n, o) Higher magnification of white rectangular region in m) at indicated axial distance; triple-view reconstruction n) vs. single view C o). p, q) Axial view along dashed line in m) , comparing triple-view reconstruction p) to single view C q) . White arrows: structures that are dim in single view but restored in triple-view. Scale bars: a) 50 μm, b-e, n-q) 10 μm, h, i, m) 20 μm, j, k) 5 μm.

Article Snippet: Heart tissue sections were incubated in 0.1% Triton X-100/PBS with 1μM of ATTO 647N-NHS ester (Sigma, 18373) and 1X NucSpot Live 488 (Biotium, 40081) at RT for 30 minutes.

Techniques: Labeling, Microscopy, Derivative Assay, Membrane, Staining, Immunolabeling

Journal: Nature Communications

Article Title: Nanovesicles loaded with a TGF-β receptor 1 inhibitor overcome immune resistance to potentiate cancer immunotherapy

doi: 10.1038/s41467-023-39035-x

Figure Lengend Snippet: a The chemical structure of the nanovesicles integrating a phospholipid prodrug of JQ1, photosensitizer pyropheophorbide a (PPa), and transforming growth factor β receptor 1 (TGFR1) inhibitor LY2157299 (LY). b Schematic illustration of the cascade drug release of the nanovesicles. Matrix metallopeptidase-2 (MMP-2) cleaved the poly(ethylene glycol) (PEG) corona and induced the LY release. PPa generated singlet oxygen upon near-infrared (NIR) laser irradiation to release JQ1-SH. c Diagram illustrating the mechanism of the nanovesicles to overcome immune resistance. The nanovesicles (ELJNV) breach the physical barrier and enhance the tumor-specific immune response upon the 671 nm laser irradiation to overcome the intrinsic immune resistance and simultaneously suppress the interferon-γ (IFN-γ)-induced inducible immune resistance in vivo. FI fluorescence imaging, PAI photoacoustic imaging, MRI nuclear magnetic resonance imaging, ROS reactive oxygen species, ECM extracellular matrix, CTLs cytotoxic T lymphocytes, α-SMA α-smooth muscle actin, PDT photodynamic therapy, ICD immunogenic cell death, PD-L1 programmed cell death 1, BRD4 bromodomain-containing protein 4, HMGB1 high mobility group box protein 1, CRT calreticulin, DC dendritic cell, CAFs cancer-associated fibroblasts, DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, JTP JQ1-thioketal (TK)-pPC, TGF-β1 transforming growth factor β1, 1 O 2 singlet oxygen, Gd 3+ gadolinium ion.

Article Snippet: Antibodies against GAPDH, calreticulin and high mobility group proteins B1 (HMGB1) were all ordered from Abcam (UK).

Techniques: Generated, Irradiation, In Vivo, Fluorescence, Imaging, Nuclear Magnetic Resonance