Journal: Advanced Science

Article Title: Therapeutic Reprogramming of Glioblastoma Phenotypic States Using Multifunctional Heparin Nanoparticles

doi: 10.1002/advs.202509590

Figure Lengend Snippet: In vivo cellular fates and molecular dynamics coordinated by HP‐NPs treatments. A) representative micrograph of BT12 patient avatar cell invasion trajectories (hVIM and yellow boxes) along the corpus callosum of the host mouse brain (PODXL, mouse microvascular endothelium, magenta and DAPI, blue). B) representative micrograph of DOX‐treated BT12 patient avatar cell invasion trajectories (hVIM and yellow boxes) along the corpus callosum of the host mouse brain (PODXL, mouse microvascular endothelium, magenta and DAPI, blue). C) representative micrograph of HP‐NP‐treated BT12 patient avatar cell invasion trajectories (hVIM and yellow boxes) at the leading edge of the tumor. D) representative micrograph of HP‐DOX‐NP‐treated BT12 patient avatar cell invasion trajectories (hVIM and yellow boxes) along the corpus callosum of the host mouse brain (PODXL, mouse microvascular endothelium, magenta and DAPI, blue). E) quantification of the tumor cell dissemination (number of invasive cells, distance from the tumor leading edge) in vehicle, DOX, HP‐NP, or HP‐DOX‐NP treated BT12 patient avatars ( n = 3 per group). F) representative micrograph of BT12 patient avatar microvascular endothelium architecture (hVIM, human tumor cells, white; PODXL, microvascular endothelium, magenta; DAPI, blue). T, intratumoral, B, healthy brain parenchyma. G) representative micrograph of DOX‐treated BT12 patient avatar microvascular endothelium architecture (hVIM, human tumor cells, white; PODXL, microvascular endothelium, magenta; DAPI, blue). T, intratumoral, B, healthy brain parenchyma. H) representative micrograph of HP‐NP‐treated BT12 patient avatar microvascular endothelium architecture (hVIM, human tumor cells, white; PODXL, microvascular endothelium, magenta; DAPI, blue). T, intratumoral, B, healthy brain parenchyma. I) representative micrograph of HP‐DOX‐NP‐treated BT12 patient avatar microvascular endothelium architecture (hVIM, human tumor cells, white; PODXL, microvascular endothelium, magenta; DAPI, blue). T, intratumoral, B, healthy brain parenchyma. J) quantification of the tumor microvascular endothelium (number of vessels per section, blood vessel area in µm 2 ) in vehicle, DOX, HP‐NP, or HP‐DOX‐NP treated BT12 patient avatars (n = 3 per group). K) Western Blot of HBEGF, human vimentin (hVim) and heat shock chaperone protein 70 (HSC70) protein expression in vehicle, DOX, HP‐NP, and HP‐DOX‐NP treated patient avatars brain extracts. L, hVimentin protein levels ratioed to the HSC70 protein loading control. M) Western Blot of epidermal growth factor receptor (EGFR), glial fibrillary acidic protein (GFAP), meteorin (MTRN) and HSC70 protein expression in vehicle, DOX, HP‐NP and HP‐DOX‐NP treated patient avatars brain extracts. Image was edited between lanes three and four to mask the protein weight ladder lane. N) HBEGF, GFAP, MTRN, and EGFR protein levels normalized to the HSC70 protein loading control and ratioed to the human tumor cell abundance (e.g., hVim) in the brain extracts. Panels E, and J: statistical significance was determined by one‐way ANOVA with non‐parametric Kruskal‐Wallis post hoc test for multiple comparisons of treatment versus vehicle.

Article Snippet: Monoclonal mouse anti‐EGFR (sc‐373746, Santa Cruz, 1:1000), monoclonal mouse anti‐HSC70 (sc‐7298, Santa Cruz, 1:1000), monoclonal mouse anti GFAP (G3893, Sigma‐Aldrich, 1:1000), polyclonal rabbit anti‐meteorin (600‐430, ThermoFisher, 1:500), monoclonal mouse anti human vimentin (C9080, Sigma‐Aldrich, 1:1000), monoclonal mouse anti GFAP (G3893, Sigma‐Aldrich, 1:1000), and monoclonal mouse anti human HBEGF (sc‐74441, Santa Cruz, 1:1000).

Techniques: In Vivo, Western Blot, Expressing, Control

Journal: Physiological Reports

Article Title: Salivary vascular growth factor responses to prolonged and interrupted sitting in young, healthy adults

doi: 10.14814/phy2.70798

Figure Lengend Snippet: Changes in salivary growth factor concentrations during prolonged sitting (PS), low‐intensity (LI), and moderate‐intensity (MI) interruption sessions. Box‐and‐whisker plots display pre‐ (Pre, 0 h) and post‐session (Post, 4 h) concentrations for (a) VEGF, (b) EGF, and (c) angiogenin. Panel (d) shows pre‐post percent change for all three biomarkers. The line within each box represents the median; whiskers indicate minimum and maximum values. VEGF decreased significantly in LI and MI but not PS; EGF showed no significant changes; and angiogenin increased significantly in all conditions. No significant differences were observed between conditions in the magnitude of pre‐post change.

Article Snippet: Samples were then analyzed using sandwich Enzyme‐Linked Immunoabsorbent Assays (ELISA) to examine EGF (Human EGF DuoSet ELISA, R&D Systems Inc.), VEGF (Human VEGF DuoSet ELISA, R&D Systems Inc.), and angiogenin (Human Angiogenin DuoSet ELISA, R&D Systems Inc.) following the manufacturer's protocol with absorbance measured at 450 nm using a Cytation 5 microplate reader (Bio‐Tek Instrumentation, VT, USA).

Techniques: Whisker Assay

Journal: Journal of Biological Chemistry

Article Title: Transactivation of the Epidermal Growth Factor Receptor Is Involved in 12-O-Tetradecanoylphorbol-13-acetate-induced Signal Transduction

doi: 10.1074/jbc.m107156200

Figure Lengend Snippet: FIG. 3. The neutralizing antibody of EGFR blocks TPA-in- duced ERK phosphorylation and AP-1 DNA binding activity. Panel A, effect of neutralizing antibodies for EGFR and HB-EGF on EGF- or TPA-induced EGFR phosphorylation. PC3 cells were cultured in a monolayer in 150-mm dishes until they reached 90% confluence and then starved in serum-free DMEM for 36 h. The cells were pre- treated with antibodies against 5 g/ml EGFR (EGFR) or 10 g/ml HB-EGF (HB-EGF) for 30 min and then treated with 600 ng/ml TPA or 10 ng/ml EGF for 3 min. The samples were immunoprecipitated by using anti-EGFR and probed with anti-phosphotyrosine (4G10) and anti-EGFR. Panel B, effect of a neutralizing antibody for EGFR on TPA- or EGF-induced ERK phosphorylation. PC3 cells (8 104/well) were cultured in a monolayer in six-well plates until they reach 90% conflu- ence, and then they were starved for 36 h in FBS-free and DMEM. The cells were pretreated with 0.5 g/ml antibody against EGFR for 30 min and then treated with 600 ng/ml TPA or 10 ng/ml EGF for 10 min. Samples were harvested with SDS sample buffer and analyzed by Western blotting with antibodies against nonphosphorylated or phos- phorylated ERKs. Panel C, effect on TPA-induced AP-1 DNA binding activity. PC3 cells were cultured in a monolayer in 10-cm dishes (1 106/dish) until they reached 90% confluence. Then, they were starved for 36 h in FBS-free and DMEM. The cells were pretreated with a 5 g/ml antibody against EGFR for 30 min followed by treatment with 600 ng/ml TPA for another 14 h. Sequence-specific AP-1 DNA binding activity was determined by gel shift analysis using a 32P-labeled oligo- nucleotide containing the AP-1 binding site.

Article Snippet: The antibodies used included rabbit polyclonal phosphorylated ERK antibodies (New England Biolabs, Beverly, MA), rabbit polyclonal EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal phosphorylated tyrosine (4G10), mouse monoclonal EGFR (neutralizing, clone LA1, Upstate Biotechnologies, Lake Placid, NY), and mouse monoclonal heparin-binding EGF (HB-EGF) (neutralizing, R&D Systems Inc., Minneapolis, MN).

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Cell Culture, Immunoprecipitation, Western Blot, Sequencing, Gel Shift, Labeling

Journal: Journal of Biological Chemistry

Article Title: Transactivation of the Epidermal Growth Factor Receptor Is Involved in 12-O-Tetradecanoylphorbol-13-acetate-induced Signal Transduction

doi: 10.1074/jbc.m107156200

Figure Lengend Snippet: FIG. 4. A neutralizing antibody for HB-EGF blocks TPA- but not EGF-induced phosphorylation of ERKs. PC3 cells (8 104/ well) were cultured in a monolayer in six-well plates until they reached 90% confluence. Then they were starved for 12 h in 0.5% FBS and DMEM. The cells were pretreated with a 10 g/ml antibody against HB-EGF (HB-EGF) for 30 min and then treated with 600 ng/ml TPA or 10 ng/ml EGF for 10 min. Samples were harvested with SDS sample buffer and analyzed by Western blotting with antibodies against non- phosphorylated or phosphorylated ERKs.

Article Snippet: The antibodies used included rabbit polyclonal phosphorylated ERK antibodies (New England Biolabs, Beverly, MA), rabbit polyclonal EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal phosphorylated tyrosine (4G10), mouse monoclonal EGFR (neutralizing, clone LA1, Upstate Biotechnologies, Lake Placid, NY), and mouse monoclonal heparin-binding EGF (HB-EGF) (neutralizing, R&D Systems Inc., Minneapolis, MN).

Techniques: Phospho-proteomics, Cell Culture, Western Blot

Journal: Nature communications

Article Title: Host interneurons mediate plasticity reactivated by embryonic inhibitory cell transplantation in mouse visual cortex.

doi: 10.1038/s41467-021-21097-4

Figure Lengend Snippet: Fig. 7 Transplant-induced ODP depends on NRG1/ErbB4 signaling. a Experimental timeline. b Intrinsic optical imaging was performed to measure ocular dominance plasticity (ODP) in adult transplant recipients around 33–35 days after transplantation (DAT). The wild-type adult recipients received either saline or soluble neuregulin 1 (NRG1) during 4 days of monocular deprivation (MD). c Examples of response amplitude before and after 4D MD in WT transplant recipients that received saline injections or were treated with NRG1 during deprivation. Scale bar = 500 µm. d Four-day MD resulted in a shift of ocular dominance index (ODI) in transplant recipients that received saline injections. Injections of exogenous NRG1 diminished transplant-induced ODI shift. Interneuron transplantation also failed to reactivate ODP in PV-cre;ErbB4flx/flx adult recipients. However, WT adults that were transplanted with cells from PV-cre;ErbB4flx/flx donor exhibited a significant shift in ODI after 4D MD. ODI is calculated as (Contra response amplitude −Ipsi response amplitude)/(Contra response amplitude + Ipsi response amplitude) (Saline n = 7 mice, red; NRG1 n = 6 mice, blue; PV-cre;ErbB4flx/flx recipients n = 6 mice, orange; PV-cre;ErbB4flx/flx donors PreMD: n = 7 mice, PostMD: n = 5 mice, yellow. Saline: **p = 0.004; NRG1: p = 0.344; PV-cre;ErbB4flx/flx

Article Snippet: To test the effect of NRG1 on the response properties of PV interneurons, some mice received subcutaneous injections of soluble NRG1 (R&D Systems, 396-HB; 0.5 μg per animal every 8 h) during MD.

Techniques: Optical Imaging, Transplantation Assay, Saline

Journal: Life Science Alliance

Article Title: HB-EGF activates EGFR to induce reactive neural stem cells in the mouse hippocampus after seizures

doi: 10.26508/lsa.202201840

Figure Lengend Snippet: HB-EGF increases early after the induction of MTLE in vivo and is secreted by NSPCs in vitro. (A) Confocal microscopy images from Nestin-GFP mice, 14dpKA showing the reduction caused by gefitinib of the MTLE-induced GCD and NSC overactivation. Scale bar, 20 μm. (B) Quantification of the volume of the GCL in saline+DMSO–, saline+ gefitinib–, KA+DMSO–, and KA+gefitinib–treated animals 14dpKA. (C) Quantification of the BrdU+ cells in saline+DMSO–, saline+ gefitinib–, KA+DMSO–, and KA+gefitinib–treated animals 14dpKA. (D) Quantification of BrdU+ NSCs, ANPs, and overall cells in the SGZ expressed as the percentage with respect to KA+DMSO animals. (E) Confocal microscopy images showing an increase in the EGFR ligand HB-EGF in the hilus and the molecular layer of Nestin-GFP mice 3dpKA. Scale bar, 20 μm. (F) Quantification of HB-EGF by ELISA at different time points during the first 3dpKA. Values are represented as the pg of HB-EGF in 100 μg of the tissue. (G) Immunofluorescence images of NSPCs in vitro showing HB-EGF expression. Scale bar, 10 μm. (H) Quantification by ELISA of HB-EGF released in vitro, expressed as the pg of the ligand per mL. Dots show individual data. Data information: for (B), Kruskal–Wallis test, * P < 0.05, * P < 0.01, and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (C), Kruskal–Wallis test, * P < 0.05, * P < 0.01, and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (D), t test, ** P < 0.01. Bars show the mean ± SEM. Dots show individual data. For (F), one-way ANOVA, * P < 0.05 and *** P < 0.001. Bars show the mean ± SEM. Dots show individual data. For (H), Kruskal–Wallis test, * P < 0.05. Bars show the mean ± SEM. Source data are available for this figure.

Article Snippet: Next, we determined the amount of HB-EGF per cell through ELISA (Cat#DY8239-05; R&D Systems).

Techniques: In Vivo, In Vitro, Confocal Microscopy, Saline, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Expressing

Journal:

Article Title: Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

doi: 10.1002/jcp.21332

Figure Lengend Snippet: DCT-induced Akt (Ser473) phosphorylation in colon cancer cells. A: To determine the time-course for bile acid-induced phosphorylation of Akt, HT-29, and H508 cells were incubated with 300 µM DCT for up to 60 min and cell extracts were immunoblotted at the times indicated to determine changes in phospho-Akt Ser473 (p-Akt). To determine the dose–response for bile acid-induced activation of Akt, cells were incubated for 5 min with increasing concentrations of DCT and cell extracts were immunoblotted for phospho-Akt Ser473. B: HT-29 and H508 cells were pre-incubated with or without inhibitors of EGFR kinase and PI3K for 30 min and then incubated for 5 min with or without DCT. Cell extracts were immunoblotted to determine changes in phospho-Akt Ser473. In these experiments, the quantity of protein added was verified by immunoblotting with antibody specific for total Akt. C: Immunofluorescence staining of H508 cells for phospho-Akt Ser473 in cells incubated for 5 min with water, 300 µM DCT, or 10 ng/ml EGF. After treatment, cells were immunostained as described in Methods Section. The first row shows results of control staining in the absence of primary (phospho-Akt) antibody. The first column shows staining with addition of secondary antibody (Alexa Fluor 555). The middle column shows Hoechst nuclear staining. The third column showing merged images of the signals obtained with the secondary antibody and Hoechst nuclear staining demonstrates cytoplasmic localization of the phospho-Akt Ser473 signal. Large arrows (middle part) indicate staining of nuclei; small arrows (right part) indicate accumulation of phosphorylated Akt in the cytoplasm of DCT-treated cells. Results are representative of three separate experiments.

Article Snippet: Antibodies against p-Akt, total Akt, p-GSK-3, p-BAD, BAD, EGFR, β-actin, and histone H2A were purchased from Cell Signaling Technology (Danvers, MA), antibodies against NF-κB p65 purchased from BD Biosciences (San Jose, CA) and antibodies against PARP p85 from Promega (Madison, WI).

Techniques: Phospho-proteomics, Incubation, Activation Assay, Western Blot, Immunofluorescence, Staining, Control

Journal:

Article Title: Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

doi: 10.1002/jcp.21332

Figure Lengend Snippet: Akt kinase activity in colon cancer cells treated with DCT alone or with DCT plus inhibitors of EGFR and PI3K. A: Dose–response and time-course for the actions of DCT on GSK substrate phosphorylation. B: Effect of adding EGFR kinase inhibitors (AG1478 and PD168398) and PI3K inhibitors (LY294002 and wortmannin). Serum-starved cells were pre-incubated with inhibitors or Me2SO (DMSO; vehicle) for 30 min followed by 5-min incubation with or without DCT. Total Akt was immunoprecipitated from HT-29 and H508 cell extracts and the in vitro kinase assay was performed using a GSK-3-paramyosin fusion protein substrate probed with phospho-GSK-3α/β Ser21/9 antibody. India Ink staining was used to determine sample loading (not shown, see Experimental Procedures Section). Results shown are representative of at least three separate experiments.

Article Snippet: Antibodies against p-Akt, total Akt, p-GSK-3, p-BAD, BAD, EGFR, β-actin, and histone H2A were purchased from Cell Signaling Technology (Danvers, MA), antibodies against NF-κB p65 purchased from BD Biosciences (San Jose, CA) and antibodies against PARP p85 from Promega (Madison, WI).

Techniques: Activity Assay, Phospho-proteomics, Incubation, Immunoprecipitation, In Vitro, Kinase Assay, Staining

Journal:

Article Title: Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

doi: 10.1002/jcp.21332

Figure Lengend Snippet: Transfection of colon cancer cells with dominant negative EGFR attenuates DCT-induced Akt phosphorylation. A: Control HT-29 cells and HT-29 cells transfected with plasmids containing wild-type (WT) and Lys721 to Met (DNM) EGFR (Cheng and Koland, 1998) were incubated for 5 min with DCT and cell extracts were immunoblotted to determine changes in phospho-Akt Ser473 (p-Akt). The quantity of protein added was verified by immunoblotting with antibodies for total Akt. Three separate experiments were performed that showed similar results. B: Graphic representation of densitometry data from immunoblots prepared as described in A. Densitometry units are arbitrary based on the value for the water control in non-transfected HT-29 cells (first lane) defined as 1. Results are expressed as mean ± SD of at least three separate experiments.

Article Snippet: Antibodies against p-Akt, total Akt, p-GSK-3, p-BAD, BAD, EGFR, β-actin, and histone H2A were purchased from Cell Signaling Technology (Danvers, MA), antibodies against NF-κB p65 purchased from BD Biosciences (San Jose, CA) and antibodies against PARP p85 from Promega (Madison, WI).

Techniques: Transfection, Dominant Negative Mutation, Phospho-proteomics, Control, Incubation, Western Blot

Journal:

Article Title: Deoxycholyltaurine Rescues Human Colon Cancer Cells From Apoptosis by Activating EGFR-Dependent PI3K/Akt Signaling

doi: 10.1002/jcp.21332

Figure Lengend Snippet: Intracellular signaling cascades activated by interaction of DCT with plasma membrane receptors. Evidence for these interactions derives from the present work and data presented in (Cheng and Raufman, 2005; Cheng et al., 2007). Stimulation of M3 muscarinic receptors (M3R) leads to activation of matrix metalloproteinase-7 (MMP-7), release of HB-EGF and activation of EGFR [blocked by AG1478, PD168393, and dominant negative (DN)-EGFR] (Cheng and Raufman, 2005; Cheng et al., 2007). Molecules indicated in yellow, activated primarily by post-EGFR ERK signaling (blocked by MEK inhibitors U0126 and PD98059), stimulate cell proliferation. In contrast, molecules indicated in green, activated primarily by post-EGFR PI3K/Akt signaling (blocked by LY294002, wortmannin and dominant negative-AKT), are largely anti-apoptotic (prosurvival). Key phosphorylations are indicated by a circled P. Both activated NF-κB and phosphorylated ERK undergo nuclear translocation where these molecules alter transcription of cell cycle and other critical genes that regulate cell proliferation and survival.

Article Snippet: Antibodies against p-Akt, total Akt, p-GSK-3, p-BAD, BAD, EGFR, β-actin, and histone H2A were purchased from Cell Signaling Technology (Danvers, MA), antibodies against NF-κB p65 purchased from BD Biosciences (San Jose, CA) and antibodies against PARP p85 from Promega (Madison, WI).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Dominant Negative Mutation, Translocation Assay