Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 3. Dual Mmp9/Mmp14 deletion does not affect Cthrc1 levels or galectin-3 expression in osteoclasts. (A) Cthrc1 (green) immunofluorescence stained with an anti-Cthrc1 polyclonal antibody (ab85739; Abcam) and F-actin (red) imaged following phalloidin staining of wild-type and DKO osteoclasts.

Article Snippet: Galectin-3 surface binding antagonist reverses functional defects in DKO osteoclasts. (A and B) Galectin-3 cleavage by Western blot using either an anti–galectin-3 full-length polyclonal antibody (Proteintech, 14979-1-AP; A) or an anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitope mapped within the N-terminal region; B), following co-incubation with 3 μg recombinant human GALECTIN-3 and activated recombinant MMP9 at molar ratios from 1:50 to 1:5 (enzyme to substrate ratio) at 37°C for 30 min.

Techniques: Expressing, Immunofluorescence, Staining

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 5. Exogenous galectin-3, but not galectin-3C, regulates osteoclast RhoA GTPase activation, sealing zone formation and bone resorptive activity. (A) Surface galectin-3 levels in wild-type osteoclasts exogenously treated with 0.1 μM galectin-3 or galectin-3C (corresponds to amino acids 108–250 containing the C-terminal carbohydrate recognition domain of galectin-3) for 30 min at 37°C (John et al., 2003; Massa et al., 1993), followed by staining with the CoraLite 488–conjugated galectin-3 polyclonal antibody (CL488-14979; Proteintech; epitopes mapped against full-length) as detected by flow cytometry. Results are representative of three independent experiments. (B) Mmp9, Mmp14, Ctsk, c-Src, and RhoA expression in wild-type osteoclasts treated with exogenous 0.1 μM galectin-3 as assessed by Western blot. Results are representative of three independent experiments. (C and D) TRAP (red), WGA-DAB, and

Article Snippet: Galectin-3 surface binding antagonist reverses functional defects in DKO osteoclasts. (A and B) Galectin-3 cleavage by Western blot using either an anti–galectin-3 full-length polyclonal antibody (Proteintech, 14979-1-AP; A) or an anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitope mapped within the N-terminal region; B), following co-incubation with 3 μg recombinant human GALECTIN-3 and activated recombinant MMP9 at molar ratios from 1:50 to 1:5 (enzyme to substrate ratio) at 37°C for 30 min.

Techniques: Activation Assay, Activity Assay, Staining, Flow Cytometry, Expressing, Western Blot

Journal: The Journal of cell biology

Article Title: Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption.

doi: 10.1083/jcb.202206121

Figure Lengend Snippet: Figure 6. Mmp9/Mmp14-dependent galectin-3 lattice remodeling controls osteoclast RhoA activation, sealing formation and bone resorptive ac- tivity. (A) Surface galectin-3 levels were determined in wild-type osteoclasts (OC) treated with control IgG, and DKO osteoclasts treated with control IgG or galectin-3 function-blocking mAb at 25 μg/ml for 2 h at 37°C, followed by staining with the CoraLite 488–conjugated galectin-3 full-length polyclonal antibody (CL488-14979; Proteintech) as detected by flow cytometry. Results are representative of three independent experiments. (B) Wild-type osteoclasts were treated with control IgG, and DKO osteoclasts were treated with control IgG or galectin-3 function-blocking mAb at 25 μg/ml for 2 h at 37°C, and RhoA activity determined upon activation with 20 ng/ml M-CSF and 30 ng/ml RANKL for 15 min. Data are presented as mean ± SEM (n = 6 biological replicates). (C and D) TRAP (red), WGA-DAB, and phalloidin staining (red) of wild-type pre-osteoclasts cultured atop bone slices treated with control IgG, and DKO pre-osteoclasts treated with control IgG or galectin-3 function-blocking mAb (Gal3 mAb; 25 μg/ml) for 3 d at 37°C (C), and the number of TRAP+ MNCs, resorption pit area, and actin ring area per cell quantified (D). Scale bar, upper and middle 100 μm, lower 20 μm. Data are presented as mean ± SEM (n = 6 biological replicates). *P < 0.05, **P < 0.01. Statistical significance was assessed using one-way ANOVA with Bonferroni correction.

Article Snippet: Galectin-3 surface binding antagonist reverses functional defects in DKO osteoclasts. (A and B) Galectin-3 cleavage by Western blot using either an anti–galectin-3 full-length polyclonal antibody (Proteintech, 14979-1-AP; A) or an anti–galectin-3 monoclonal antibody (ab2785; Abcam; epitope mapped within the N-terminal region; B), following co-incubation with 3 μg recombinant human GALECTIN-3 and activated recombinant MMP9 at molar ratios from 1:50 to 1:5 (enzyme to substrate ratio) at 37°C for 30 min.

Techniques: Activation Assay, Control, Blocking Assay, Staining, Flow Cytometry, Activity Assay, Cell Culture

Journal: Nature Communications

Article Title: Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

doi: 10.1038/s41467-019-08921-8

Figure Lengend Snippet: Fyn phosphorylates 6-phosphogluconate dehydrogenase (6PGD) at Y481. a HEK293T/epidermal growth factor receptor (EGFR) cells were pretreated with Afatinib (1 μM), Ruxolitinib (1 μM), Saracatinib (1 μM), or Amuvatinib (10 μM) for 3 h and then treated with EGF (100 ng ml -1 ) for 30 min. b HEK293T/EGFR cells stably expressing Flag-6PGD WT or Y481F were transiently co-transfected with or without HA-Src, HA-Fyn as indicated. c HEK293T/EGFR cells were transiently co-transfected with or without Flag-6PGD and a kinase-dead HA-Fyn (KD) mutant as indicated. Cells were treated with or without EGF (100 ng ml -1 ) for 30 min. d U87/EGFR cells were infected with the lentivirus expressing shNT or two shRNA sequences against Fyn (shFyn-1 and shFyn-2). These cells were then treated with or without EGF (100 ng ml -1 ) for 30 min as indicated. e U87/EGFR cells were transiently co-transfected with HA-Fyn and Flag-6PGD as indicated. Cells were treated with or without EGF (100 ng ml -1 ) for 30 min. f In vitro kinase assays were carried out with bacterial purified recombinant His-6PGD WT or Y481F and commercial purchased recombinant GST-Fyn. g HA-SBP (HB) tagged Fyn WT or kinase-dead HB-Fyn (KD) mutant was immunoprecipitated from U87/EGFR cells pretreated with EGF (100 ng ml -1 ) for 30 min and then used for in vitro kinase assays with bacterial purified His-6PGD. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. Source data are provided as a Source Data file

Article Snippet: Recombinant human Fyn (active) (F15-10G-10) and EGFR (active) (E10-11G-10) were purchased from SignalChem (British Columbia, Canada).

Techniques: Stable Transfection, Expressing, Transfection, Mutagenesis, Infection, shRNA, In Vitro, Purification, Recombinant, Immunoprecipitation, Western Blot

Journal: Nature Communications

Article Title: Tyrosine phosphorylation activates 6-phosphogluconate dehydrogenase and promotes tumor growth and radiation resistance

doi: 10.1038/s41467-019-08921-8

Figure Lengend Snippet: 6-Phosphogluconate dehydrogenase (6PGD) pY481 enhances NADP + binding affinity of 6PGD. a U87/epidermal growth factor receptor (EGFR) cells stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml -1 ) for 30 min. Cell lysates were incubated with Cibacron blue beads mimicking NADP + for a pulldown assay. b In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without active recombinant Fyn. ITC assays were performed with pulldown 6PGD variants (0.05 mM) and NADP + (1 mM) (top panel). His peptides were used as negative control. Statistical analyses of K d values of 6PGD variants for NADP + were presented (bottom panel). c , d In vitro kinase assays were performed by incubating bacterial purified recombinant His-6PGD WT or Y481F with or without recombinant active Fyn. K m ( c ) and k cat ( d ) of 6PGD variants were determined. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. b – d Data represent the mean ± SD of three independent experiments. Student’s t -test (unpaired, two tailed), ** p < 0.01; n.s. not significant. Source data are provided as a Source Data file

Article Snippet: Recombinant human Fyn (active) (F15-10G-10) and EGFR (active) (E10-11G-10) were purchased from SignalChem (British Columbia, Canada).

Techniques: Binding Assay, Stable Transfection, Expressing, Incubation, In Vitro, Purification, Recombinant, Negative Control, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: Neuro-Oncology

Article Title: Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish

doi: 10.1093/neuonc/nos387

Figure Lengend Snippet: Targeted expression of transgenes in embryos. (A) Transgenesis strategy. Gal4-UAS system allows targeted expression of transgenes. (B) Inverted fluorescence images (upper column, dorsal views; lower column, lateral views). Transgenic GFP expression is spatiotemporally restricted to the Ptf1a domain. Exocrine pancreatic expression of DARac1 disturbs the posterior expansion of the exocrine pancreas. The morphology of the hindbrain is not altered by either DAAkt1 or DARac1 expression. (C) Whole-mount ISH at 2 dpf (dorsal views) and 4 dpf (lateral views). (D) Confocal images showing membrane localization of GFP fused with DARac1. Abbreviations: H, hindbrain; R, retina; P, exocrine pancreas. Bars, 20 μm.

Article Snippet: 28 , 29 Human DAAkt1 (1036 pcDNA3 Myr-HA-Akt1; myristoylated AKT1 targeted to the membrane independently of PtdIns-3,4,5-P3 and referred to as dominant-active human AKT1) was purchased from Addgene (Cambridge, MA), and human Rac1 cDNA was purchased from Open Biosystems (Huntsville, AL). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 Targeted expression of transgenes in embryos. (A) Transgenesis strategy.

Techniques: Expressing, Fluorescence, Transgenic Assay, Membrane

Journal: Neuro-Oncology

Article Title: Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish

doi: 10.1093/neuonc/nos387

Figure Lengend Snippet: DAAkt1-induced gliomas. (A) Gross and histologic findings. (a,b) Glioma-bearing 3-month-old Ptf1aGal4/UASGFP-UASDAAkt1 zebrafish with bent body and visible bumps at the head, showing strong GFP expression on merged bright and fluorescence images (inlets). (c) Hematoxylin and eosin stains of cerebellum in control zebrafish. (d–i) Hematoxylin and eosin stains of gliomas. (d and e) Small tumors (boundaries by red arrowheads) at periventricular area showing invasion into 4th ventricle. (f) A large glioma replacing almost the whole cerebellum. (g) A large glioma showing invasion into the midbrain. (h) A high-grade glioma showing increased vascularity (black arrowhead). (i) A high-grade glioma showing necrosis (red arrowhead). (j) IHC for GFAP showing robust expression. (k) IHC for Akt1 on a small tumor invading 4th ventricle showing expression at tumor. (l) ISH for DAAkt1 showing stronger expression at the hypercellular area. (B) Increased proliferation in DAAkt1-induced gliomas. IHC indicates highly frequent positivity to PCNA, which is even higher in high-grade tumor. Tumors occasionally show positive staining to PHH3, a mitotic marker. BrdU labeling also reveals frequently positive cells. (C) Proliferation analyses in the pre-neoplastic cerebellum of 2-month-old zebrafish. Reactivity at the intestinal cells is used as an internal positive control (Inlets). Cells at the ventricular lining and the intermediate layer frequently reveal immunoreactivity to proliferative markers. The non–tumor-bearing cerebellum of DAAkt1-expressing zebrafish is not associated with increased proliferation. Average numbers of positive cells counted at the periventricular are 4.5 ± 2.2 and 4.7 ± 2.6 for PHH3 and 18.7 ± 6.8 and 19.9 ± 6.9 for BrdU in Ptf1aGal4/UASGFP and Ptf1aGal4/UASGFP-UASDAAkt1 zebrafish, respectively, which are not significantly different. Bars, 50 μm. (D) Activation of Akt downstream components in nonneoplastic DAAkt1-expressing cerebellum. IHC analyses reveal slightly increased expression of phospho-mTOR, -RS6K, and -4EBP1 in cells at the ventricular and periventricular zone.

Article Snippet: 28 , 29 Human DAAkt1 (1036 pcDNA3 Myr-HA-Akt1; myristoylated AKT1 targeted to the membrane independently of PtdIns-3,4,5-P3 and referred to as dominant-active human AKT1) was purchased from Addgene (Cambridge, MA), and human Rac1 cDNA was purchased from Open Biosystems (Huntsville, AL). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 Targeted expression of transgenes in embryos. (A) Transgenesis strategy.

Techniques: Expressing, Fluorescence, Control, Staining, Marker, Labeling, Positive Control, Activation Assay

Journal: Neuro-Oncology

Article Title: Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish

doi: 10.1093/neuonc/nos387

Figure Lengend Snippet: DARac1 accelerates DAAkt1-induced glioma. (A) Two-month-old zebrafish showing bumps at the head. Inlets are merged bright field and fluorescence images. (B) Histologic findings of gliomas in Ptf1aGal4/UASGFP-UASDAAkt1/UASGFP-DARac1 zebrafish. (a-g) Hematoxylin and eosin staining. Tumors frequently reveal heterogeneous grade of glioma showing a hypercellular nest within the tumor (red arrowheads) and increased vascularity (black arrowheads). (g and h) IHC and ISH for transgenes in a high-grade glioma. Inlets of (g) and (h) are IHC for Akt1 and ISH for DARac1, respectively. (i and j) ISH for DARac1. Cells at the invasion front show stronger expression of DARac1. Arrows indicate the direction of invasion. (k and l) Proliferation analyses reveal increased positivity to PCNA and PHH3, especially at the hypercellular areas, which accompanies a more robust expression of DARac1 on ISH (inlet). (C) Histologic grade of gliomas. Coexpression of DARac1 increased not only the tumor incidence but also the histologic grades of gliomas (D) Tumor incidence and mortality. 112 zebrafish from each Ptf1aGal4/UASGFP-UASDAAkt1 and Ptf1aGal4/UASGFP-UASDAAkt1/UASGFP-DARac1 line were followed. Tumor incidence was estimated by body bending, which preceded the appearance of an obvious bump in the head. The incidence rates of glioma in Ptf1aGal4/UASGFP-UASDAAkt1 and Ptf1aGal4/UASGFP-UASDAAkt1/UASGFP-DARac1 zebrafish are 14.3% and 44.4% at 3 months, 36.6% and 62.0% at 6 months, and 49.1% and 73.2% at 9 months, respectively. The mortality rates are also increased by the coexpression of DARac1: 0.9% vs. 22.2% at 3 months, 15.2% vs. 50.9% at 6 months, and 21.4% vs. 60.2% at 9 months. The tumor-free survival rates are much lower in the Ptf1aGal4/UASGFP-UASDAAkt1/UASGFP-DARac1 line. Bars, 50 μm.

Article Snippet: 28 , 29 Human DAAkt1 (1036 pcDNA3 Myr-HA-Akt1; myristoylated AKT1 targeted to the membrane independently of PtdIns-3,4,5-P3 and referred to as dominant-active human AKT1) was purchased from Addgene (Cambridge, MA), and human Rac1 cDNA was purchased from Open Biosystems (Huntsville, AL). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 Targeted expression of transgenes in embryos. (A) Transgenesis strategy.

Techniques: Fluorescence, Staining, Expressing

Journal: Neuro-Oncology

Article Title: Glioma is formed by active Akt1 alone and promoted by active Rac1 in transgenic zebrafish

doi: 10.1093/neuonc/nos387

Figure Lengend Snippet: Differential genes involved in gliomagenesis. (A) Real-time RT-PCR using dissected glioma and control cerebellum shows changes in transcripts. Survivin1 and p21 are upregulated in pre-neoplastic cerebellum by DAAkt1 expression. DAAkt1-induced glioma shows upregulation of cyclin D1, p21, Survivin1, 2, and snail1a. Coexpression of DARac1 reveals further upregulation of survivin2 and snail1a, and downregulation of E-cadherin. *P< 0.05. Mann-Whitney U test was used for statistical differences. Electrophoretic (B) and Western blot (C) images confirm quantitative RT-PCR findings. C, control; A, Ptf1aGal4/UASGFP-UASDAAkt1 without tumor; A(T), Ptf1aGal4/UASGFP-UASDAAkt1 with tumor; Ptf1aGal4/UASGFP-UASDAAkt1/UASGFP-DARac1 with tumor. (D) Expression analyses of differential genes by IHC (c, g, i, k, l, n, p, and q) or ISH (a, b, d, e, f, h, j, m, o, and r). Dotted red lines indicate tumor boundaries. Gliomas coexpressing DAAkt1 and DARac1 show stronger expression of survivin2, snail1a, β-catenin, and cyclin D1, especially at the hypercellular area (red arrowheads). Expression of pan-cadherin and E-cadherin is downregulated in glioma coexpressing DARac1, especially at the higher-grade area (* in p). Bars, 50 μm.

Article Snippet: 28 , 29 Human DAAkt1 (1036 pcDNA3 Myr-HA-Akt1; myristoylated AKT1 targeted to the membrane independently of PtdIns-3,4,5-P3 and referred to as dominant-active human AKT1) was purchased from Addgene (Cambridge, MA), and human Rac1 cDNA was purchased from Open Biosystems (Huntsville, AL). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Fig. 1. caption a7 Targeted expression of transgenes in embryos. (A) Transgenesis strategy.

Techniques: Quantitative RT-PCR, Control, Expressing, MANN-WHITNEY, Western Blot