Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 1. HMGB1 binds to RB. (A) endogenous association of HMGB1 and RB. Nuclear extracts were prepared from MCF-7 and BT-549 cells and subjected to IP/Western blotting analyses. A mouse IgG IP from MCF-7 cells was used as the negative control. Antibodies used were: HMGB1 IP (a rabbit polyclonal antibody) and HMGB1 Western Blot (a mouse monoclonal antibody), RB IP (M-153), and RB Western Blot (C-15). Representative results are shown from 3 independent experiments. (B) In vitro interaction of HMGB1 and RB. GST capture assay was performed. 35S-methionine-labeled proteins were prepared by in vitro transcription and translation using the T3 promoter of mammalian expression pCMV-Tag2B vector that was used for cloning the full-length of wtHMGB1 or HMGB1-RXRXH cDNA. GST RB fusion proteins were generated from a RB cDNA cloned into a GST pGEX vector, expressed in Escheria coli, and purified by affinity chromatography (left panel). Input lanes show 10% IVT wtHMGB1 or 10% IVT HMGB1-RXRXH product used in the assay.

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Western Blot, Negative Control, In Vitro, Labeling, Expressing, Plasmid Preparation, Cloning, Generated, Clone Assay, Purification, Affinity Chromatography

Journal: Acta pharmacologica Sinica

Article Title: Growth suppression and radiosensitivity increase by HMGB1 in breast cancer.

doi: 10.1111/j.1745-7254.2007.00669.x

Figure Lengend Snippet: Figure 3. HMGB1 causes RB-dependent suppression of cell proliferation. (A) time-course of recombinant adenovirus-directed expression of HMGB1. MCF-7 cells were infected with Ad5wtHMGB1 at a MOI of 100 pfu/cell. Protein extracts were prepared at the indicated times following infection; 50 µg protein from each lysate was resolved by SDS-PAGE and probed with a mouse monoclonal anti-HMGB1 antibody. As an internal control for the amount of protein loaded, the same filter was also immunoblotted with a polyclonal α-actin antibody (I-19). Immunoreactive proteins were visualized by ECL. (B) adenovirus-mediated expression of the HMGB1 gene inhibits MCF-7, but not BT-549 breast cancer cell proliferation. Cells were seeded at 3×104 cells/well in 6-well plates 1 d before infection with Ad5-wtHMGB1, Ad5-HMGB1- RXRXH, or Ad5-lacZ (a control) at a MOI of pfu/cell. At the indicated times, the cells in 10 wells were collected and counted by a hemocytometer. SEM from 10 wells in 3 independent experiments were less than 10%. Values for Ad5-wtHMGB1 were significantly different from those for Ad5-HMGB1–RXRXH and Ad-lacZ alone in MCF-7 (P<0.001, two-tail Student’s t-test).

Article Snippet: Equal aliquots of protein extract (100 μg/lane) were electrophoresed on SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), and blotted with primary antibodies: a mouse monoclonal anti-HMGB1 antibody (R&D Systems, Minneapolis, MN, USA); a rabbit polyclonal anti-RB antibody (C-15, Santa Cruz Biotechnology, USA) or a mixture of a rabbit polyclonal anti-PRAP antibody (P7607, Sigma, USA) plus a rabbit polyclonal anti-PRAP antibody (214/215, cleavage site specific antibody, Sigma, USA).

Techniques: Recombinant, Expressing, Infection, SDS Page, Control

Journal: Antioxidants & Redox Signaling

Article Title: Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena sp. PCC 7120

doi: 10.1089/ars.2014.6175

Figure Lengend Snippet: Multiple sequence alignment of Fur proteins present in different bacteria. The abbreviations correspond to the following microorganisms: Ma: Microcystis aeruginosa PCC 7806, Sy: Synechocystis PCC 6803, An: Anabaena PCC 7120, Hp: Helicobacter pylori 26695, Mt: Mycobacterium tuberculosis H37Rv, Lm: Listeria monocytogenes EGDe, Sa: Staphylococcus aureus ATCC BAA-39, Ef: Enterococcus faecalis ATCC 29212, Cld: Clostridium difficile ATCC 43255, Cod: Corynebacterium diphtheriae ATCC 13812, Ec: Escherichia coli BL21, Se: Salmonella enterica Typhimurium LT2, Kp: Klebsiella pneumoniae ATCC 43816, Pm: Proteus mirabilis HI 4320, Vc: Vibrio cholerae CECT512, Lp: Legionella pneumophila Philadelphia-1, Pa: Pseudomonas aeruginosa ATCC 27853.

Article Snippet: The abbreviations correspond to the following microorganisms: Ma: Microcystis aeruginosa PCC 7806, Sy: Synechocystis PCC 6803, An: Anabaena PCC 7120, Hp: Helicobacter pylori 26695, Mt: Mycobacterium tuberculosis H37Rv, Lm: Listeria monocytogenes EGDe, Sa: Staphylococcus aureus ATCC BAA-39, Ef: Enterococcus faecalis ATCC 29212 , Cld: Clostridium difficile ATCC 43255, Cod: Corynebacterium diphtheriae ATCC 13812, Ec: Escherichia coli BL21, Se: Salmonella enterica Typhimurium LT2, Kp: Klebsiella pneumoniae ATCC 43816, Pm: Proteus mirabilis HI 4320, Vc: Vibrio cholerae CECT512, Lp: Legionella pneumophila Philadelphia-1, Pa: Pseudomonas aeruginosa ATCC 27853.

Techniques: Sequencing, Bacteria

Journal: Biology

Article Title: Omics Strategies in Current Advancements of Infectious Fish Disease Management

doi: 10.3390/biology10111086

Figure Lengend Snippet: Compilation of studies on fish immune response using various omics approaches.

Article Snippet: Koi ( Cyprinus carpio ) , Cyprinid herpesvirus 3 (CyHV3) , Spleen , Illumina HiSeq 2500 , [ ] .

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Liquid Chromatography, Mass Spectrometry, Chromatography, Nuclear Magnetic Resonance, Spectroscopy, Gas Chromatography-Mass Spectrometry, Fluorescence In Situ Hybridization

Journal: Cell reports

Article Title: Extracellular-vesicle-packaged S100A11 from osteosarcoma cells mediates lung premetastatic niche formation by recruiting gMDSCs.

doi: 10.1016/j.celrep.2024.113751

Figure Lengend Snippet: Figure 6. S100A11 transmitted by EVs activates JAK2-STAT3 signaling pathway in macrophages (A) The JAK-STAT signaling pathway was significantly enriched in BMDMs treated with K7M2-EVs, as analyzed by GSEA. (B) BMDMs were treated with PBS-ctrl, K7-EVs, K7M2-EVs, and IL-4 for 24 h or K7M2-EVs for the indicated times. Phosphorylation of JAK2 and STAT3 and their corresponding total levels were detected by immunoblot. Three independent experiments were performed. (C) Phosphorylation of JAK2, STAT3, AKT, and their corresponding total levels in K7M2-EV-stimulated BMDMs with different concentrations of the JAK2 inhibitor fedratinib (0, 250, and 500 nM). Three independent experiments were performed. (D) RT-qPCR analyses of the indicated genes in BMDMs treated with K7M2-Exo with fedratinib (250 nM) or BP-1-102 (1 mM) for 24 h (n = 3). (E) Quantification of CXCL2 in the supernatants of BMDMs treated as described in (D). (F) In vitro chemotaxis assay of gMDSCs using conditioned media from (D). (G) Heatmaps showing the top 10 upregulated and downregulated protein cargos in K7M2-EVs compared with K7-EVs, as detected by liquid chromatography- MS (n = 3 per group). A red triangle marks the upregulated protein S100A11 in K7M2-EVs. (H) Expression of S100A11 and the indicated EV markers in cell lines and their corresponding EVs detected by WB. GAPDH was used as a control for total cell lysate. (I) WB showing the knockdown efficacy of shS100a11 in K7M2 cells and overexpression of S100A11 in K7 cells. (J) Phosphorylation of JAK2 and STAT3 and their corresponding total levels in BMDMs treated with EVs from K7M2-shCtrl, K7M2-shS100a11, K7-vector, or K7- S100a11 cell lines for 24 h (K) ELISA detection of CXCL2 in the supernatants of BMDMs treated as described in (D) (n = 3). (L) ELISA detection of CXCL2 in the supernatants of BMDMs treated with PBS-ctrl and EVs from K7-vector or K7-S100a11 for 24 h (n = 3). (M) Bioluminescence images and quantification of lung metastasis 4 weeks after orthotopic implantation of K7M2-shCtrl and K7M2-shS100a11 cells (n = 5 mice per group).

Article Snippet: For treatment with SB225002, BP-1-102 or fedratinib (Selleck, China), the indicated doses of reagents were added to the upper chamber.

Techniques: Phospho-proteomics, Western Blot, Quantitative RT-PCR, In Vitro, Chemotaxis Assay, Liquid Chromatography, Expressing, Control, Knockdown, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: PKCα-dependent interaction of otoferlin and calbindin: evidence for regulation of endocytosis in inner hair cells

doi: 10.1101/779520

Figure Lengend Snippet: A, B PLA for otoferlin and PKCα performed on WT P15 IHCs at rest, after strong stimulation for 1 (Stim 1’) and 5 minutes (Stim 5’), and after a 5-minute recovery period post stimulation (Recov 5’ (Stim 1’)). (A) High magnification views of representative PLAs: calbindin (blue) was used as IHC marker; PLA channel is depicted with an intensity-coded lookup table with warmer colors representing higher pixel intensities. (B) Average otoferlin/PKCα PLA puncta fluorescence intensity per cell for all conditions, normalized to the resting condition. Individual cells are depicted with lighter colors and open symbols. See Appendix Figure S2A and C for control PLAs. C Representative immunoblot showing results of anti-HA and anti-GFP co-immunoprecipitation from lysates of HEK293 cells co-transfected with otoferlin-HA and GFP-PKCα. Samples were probed for HA and GFP. Upper panel depicts constructs used in binding assays. D Representative immunoblot showing results from pull-down assay from organs of Corti loaded onto anti-HA beads with previously bound otoferlin-HA expressed in HEK293 cells. Samples were probed for HA and PKCα. Upper left panel depicts constructs used in binding assays. Upper right panel depicts scheme of the assay. E Schematic representation of the in vitro phosphorylation assay and subsequent mass spectrometry analysis to assess PKCα-induced phosphosites on otoferlin. Alkaline phosphatase (AP) was used to remove any residual phosphate groups and obtain dephosphorylated otoferlin. After incubation with AP + PKCα or with AP only, samples were loaded onto a SDS-PAGE gel, otoferlin bands were excised, digested and analyzed by LC-MS/MS. For annotated MS/MS spectra of detected phosphosites, LC-MS/MS profiling of phosphopeptides and mapping of sites in the otoferlin sequence refer to Appendix Figures S3-S9 . F Position of the phosphorylation sites in the otoferlin sequence (mouse, isoform 4, NP_001300696.1) determined by LC-MS/MS. For mapping of sites in the otoferlin sequence refer to Appendix Figure S10 . Data information: In (A), maximum intensity projections of confocal optical sections. Scale bars: 5 μm. In (B), data are displayed as mean ± s.e.m.; ***P≤0.001 (Kruskal-Wallis test followed by Dunn’s multiple comparison test); mean averages, sample size and statistical analysis are detailed in Appendix Table S1 . In (A-B), only puncta inside the cells were considered for quantification purposes; quantification was performed in Imaris as described in Materials and Methods . PLA, proximity ligation assay. IHC, inner hair cell. Otof, otoferlin.

Article Snippet: Membranes were probed with primary antibodies mouse anti-HA (#MMS-101P, Covance, 1:1000) and mouse anti-GFP (#600-301-215, Rockland, 1:1000) followed by incubation with secondary antibody goat anti-mouse IgG-HRP (#115-035-146, Jackson ImmunoResearch, 1:2000).

Techniques: Marker, Fluorescence, Western Blot, Immunoprecipitation, Transfection, Construct, Binding Assay, Pull Down Assay, In Vitro, Phosphorylation Assay, Mass Spectrometry, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Sequencing, Proximity Ligation Assay