Journal: PLoS ONE

Article Title: Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

doi: 10.1371/journal.pone.0070908

Figure Lengend Snippet: Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus. Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) and stromal nerve definitely decreased from day1 (F; day 1, G; day 7, H; day 14) and after trigeminal axotomy. The nerve of the contralateral eye decreased from day 1 (J; day 1, K; day 7, L; day 14).

Article Snippet: Corneas were then stained with monoclonal NL637-conjugated anti-β-III tubulin antibody (anti-Neuron-specific β-III Tubulin-NL637, R&D systems Inc. Minneapolis, MN; dilution of 1∶100) at 4C degree overnight.

Techniques: Staining

Journal: PLoS ONE

Article Title: Bilateral Nerve Alterations in a Unilateral Experimental Neurotrophic Keratopathy Model: A Lateral Conjunctival Approach for Trigeminal Axotomy

doi: 10.1371/journal.pone.0070908

Figure Lengend Snippet: Representative histological photographs of corneal nerves stained with anti-βIII tubulin FITC-conjugated antibody. Normal subbasal (A) and stromal (E) nerve plexus.Subbasal nerve plexus completely disappeared (B; day 1, C; day 7, D; day 14) (A) and stromal nerve definitely decreased (B) from day 1 (F; day 1, G; day 7, H; day 14) after trigeminal axotomy. The nerve of the contralateral eye did not change (J; day 1, K; day 7, L; day 14).

Article Snippet: Corneas were then stained with monoclonal NL637-conjugated anti-β-III tubulin antibody (anti-Neuron-specific β-III Tubulin-NL637, R&D systems Inc. Minneapolis, MN; dilution of 1∶100) at 4C degree overnight.

Techniques: Staining

Journal: Toxicological Sciences

Article Title: Editor’s Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics

doi: 10.1093/toxsci/kfw254

Figure Lengend Snippet: Tuj-1 and vimentin as specific markers for neuronal and non-neuronal DRG cells in culture, respectively. A, Full-length image of a Wes™ Simple Western blot for Tuj-1, vimentin, and GAPDH). The whole cell lysates were collected from untreated cells on Day 8 (72 h time point); lysates loaded in each capillary at 80 pg protein and probed with anti-tuj-1 (MO15013 1:50, plus anti-GAPDH 1:100) or anti-vimentin (AB8069 1:1000, plus anti-GAPDH 1:100); Tuj-1, Vimentin and GAPDH were detected at 55, 58 and 42 kDa, respectively. B, Tuj-1 and vimentin expression normalized to GAPDH (mean ± SEM, n = 3–4). C, A representative field of four channels; merged images taken with 20× objective from a vehicle control well. Neurons, neurites and non-neuronal cells, processes are stained positive for Tuj-1 (in red) and Vimentin (in green), respectively; Closed arrows point to the neuronal cell body (Tuj-1 in red) and nuclei (NeuN in white); Open arrows point to running of a single thread of neurite and process in close contact; Stars denote cells with a unique morphology distinctive from Schwann cells.

Article Snippet: The primary antibodies were diluted with antibody diluent (ProteinSimple) at 1:50 for Tuj-1 (Neuromics, MO15013) and SOX2 (Abcam, ab137385), 1:1000 for vimentin (Abcam, ab8069), 1:25 for glutamine synthetase (EMD Millipore, MAB302).

Techniques: Western Blot, Expressing, Staining

Journal: Toxicological Sciences

Article Title: Editor’s Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics

doi: 10.1093/toxsci/kfw254

Figure Lengend Snippet: Image processing for multiparametric analysis with IN Cell Workstation software. A and B, Fluorescence images merged with NeuN and Tuj-1 channels or DAPI and vimentin channels, respectively; C and D, Show segmentation of nuclei (circled in blue lines), cell bodies (circled in green lines) and neurites or non-neuronal cell processes (outlined in yellow lines). Arrows in each image point to identical nuclei of neuronal cells (A and C) or non-neuronal cells. Scale bar = 40 µm.

Article Snippet: The primary antibodies were diluted with antibody diluent (ProteinSimple) at 1:50 for Tuj-1 (Neuromics, MO15013) and SOX2 (Abcam, ab137385), 1:1000 for vimentin (Abcam, ab8069), 1:25 for glutamine synthetase (EMD Millipore, MAB302).

Techniques: Software, Fluorescence

Journal: iScience

Article Title: Downregulation of neurodevelopmental gene expression in iPSC-derived cerebral organoids upon infection by human cytomegalovirus

doi: 10.1016/j.isci.2022.104098

Figure Lengend Snippet:

Article Snippet: Chicken polyclonal anti Tuj1 , GeneTex , GTX85469.

Techniques: Virus, Recombinant, Isolation, Control, Reverse Transcription, SYBR Green Assay, Software

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: Apigenin induces phosphorylation of Hsp27 and activation of p38 and ERK-MAPK and PKC δ . Lysates from THP-1 cells treated with 50 μ M apigenin for different lengths of time were analyzed by immunobloting with ( a ) anti-phospho-Hsp27-pS78, anti-phospho-Hsp27-pS82, anti-phospho-Hsp27-pS15, anti-total-Hsp27, and anti-Tubulin antibodies. ( b ) Immunoblots with anti-phospho-p38, anti-phospho-ERK, anti-total-p38, anti-total-ERK, and anti- β -Tubulin antibodies. ( c ) PKC δ kinase activity was determined by in vitro kinase assays. Total cell lysates were immunoprecipitated (IP) with anti-PKC δ antibodies or an isotype control (IgG) followed by in vitro kinase assay in the presence of ( γ - 32 P)ATP and H2B as substrate. Phosphorylated kinase products were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Phospholabeled proteins were visualized by autoradiography. Immunoblots of the same membrane with PKC δ served as loading control. Results are a representative of four independent experiments

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques: Activation Assay, Western Blot, Activity Assay, In Vitro, Immunoprecipitation, Kinase Assay, SDS Page, Autoradiography

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: Apigenin-induced ‘early' Hsp27 phosphorylation is dependent on p38 activity. Lysates from THP-1 cells treated with the diluent alone (lane 1), pretreated with 10 μ M p38 inhibitor SB203580 or diluent (lanes 3 and 2) before the addition of 50 μ M apigenin for 15 min following or treated with the p38 inhibitor SB203580, and diluent (lane 4) were immunoblotted with anti-phospho-Hsp27-pS78, anti-phospho-Hsp27-pS82, anti-phospho-p38, anti-total-Hsp27, anti-total-p38 antibodies. The same membrane was immunoblotted with anti-Tubulin antibodies as loading control. Results are a representative of four independent experiments

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques: Activity Assay

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: Apigenin-induced ‘late' Hsp27 phosphorylation is ERK-independent. Lysates from THP-1 cells were treated with diluent (lane 1), or pretreated with diluent or 25 μ M PD98059 before the addition of 50 μ M apigenin for 6 h (lanes 2 and 3, respectively) and immunoblotted with anti-phospho-Hsp27-pS78, anti-phospho-Hsp27-pS82, anti-phospho-Hsp27-pS15, anti-phospho-ERK, anti-total-Hsp27, anti-total-ERK antibodies. The same membrane was immunoblotted with anti- β -Tubulin antibodies as loading control. Results are a representative of four independent experiments

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques:

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: ‘Late' Hsp27 phosphorylation induced by apigenin is modulated by p38-MAPK and PKC δ . Lysates from THP-1 cells treated with 50 μ M apigenin for 6 h and diluent or following 1 h pretreatment with 10 μ M SB203580, 15 μ M rottlerin, or both. Treatments marked with (−) denote addition of diluent. ( a ) Immunoblots with anti-phospho-Hsp27-pS78 and anti-total-Hsp27 antibodies. ( b ) Immunoblots with anti-phospho-Hsp27-pS82 and anti-total-Hsp27 antibodies. ( c ) Immunoblots with anti-phospho-Hsp27-pS15 and anti-total-Hsp27 antibodies. Densitometry data normalized by the loading control are represented as mean±S.E.M. ( N =5, * P ⩽0.05 and ** P ⩽0.001, NS denotes non-statistical significance)

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: PKC δ and p38-MAPK regulate the ‘late' phosphorylation of Hsp27 induced by apigenin. Silencing of p38 and PKC δ inhibits apigenin-induced-Hsp27 phosphorylation. ( a ) Immunoblots of lysates from THP-1 cells non-transfected (NT) or transfected with siRNA-Control or siRNA-p38 were treated with diluent (−) or treated for 6 h with 50 μ M apigenin (+). ( b ) Immunoblots of lysates from THP-1 cells non-transfected (NT) or transfected with siRNA-Control or siRNA-PKC δ and treated with diluent (−) or treated for 6 h with 50 μ M apigenin (+). Results are a representative of three independent experiments

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques: Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: Hsp27 phosphorylation increases apigenin-induced apoptosis. THP-1 cells transiently transfected with empty vector or Hsp27-WT, Hsp27-A, or Hsp27-D were treated with diluent control for 9 h (NT) or with 50 μ M apigenin for different times. ( a ) Percentage of apoptotic cells was determined by Annexin V/7-AAD staining. ( b ) Active caspase-3 was determined in the same cells by FACS. ( c ) Lysates from cells obtained in ( a ) were used to determine caspase-3 activity by the DEVD-AFC assay. ( d ) Lysates from cells obtained in ( a ) were used to determine inactive full-length (FL-casp-3) and cleaved caspase-3 (cleaved casp-3) by immunoblotting. Values shown in ( a ), ( b ), and ( c ) represent mean±S.E.M., N =4, * P ⩽0.01

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques: Transfection, Plasmid Preparation, Staining, Activity Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein–27

doi: 10.1038/cddis.2010.41

Figure Lengend Snippet: Working model of regulation of Hsp27 phosphorylation during apigenin-induced apoptosis

Article Snippet: Phospho-Hsp27-pS15 (SPA-525), phospho-Hsp27-pS78 (SPA-523), phospho-Hsp27-pS82 (SPA-524), and total Hsp27 (SPA-803) antibodies were obtained from Stressgen (British Columbia, Canada).

Techniques: