Journal: Cerebral Cortex (New York, NY)

Article Title: ARX Regulates Cortical Intermediate Progenitor Cell Expansion and Upper Layer Neuron Formation Through Repression of Cdkn1c

doi: 10.1093/cercor/bht222

Figure Lengend Snippet: The cyclin-dependent kinase inhibitor (CKi) Cdkn1c is ectopically expressed in Arx KO cortical VZ and SVZ and it is a direct transcriptional target of ARX. In situ hybridizations for Cdkn1c on telencephalic coronal sections at different embryonic stages (E12.5–E14.5) of control (left) and Arx KO (right) brain tissues; in situ hybridizations for Arx on control coronal sections at the corresponding developmental stages are also showed. (A–C) At E12.5 Cdkn1c transcript is detected in the neo-formed SVZ in control brains (arrowheads, A′); differently, in Arx KO, it is significantly upregulated in both VZ and SVZ (arrowheads, B′), where Arx is normally expressed at this stage (C). Similarly, Cdkn1c is found upregulated in Arx KO VZ and SVZ at E14.5 (arrowheads, E′) whereas in the relative control sections it is still barely detected in the SVZ (arrowheads, D′). (G) A 5-kb genomic region upstream to the murine Cdkn1c gene locus was screened for the presence of paired homeodomain binding sites: a palyndromic sequence 5′-ATTAGCATAAT-3′, highly conserved in mouse, rat, human, and orangutan, was found between −71/−81 bp before the Cdkn1c transcription start site (+1). (H) A <500 bp (−549/−68) genomic region containing the putative ARX binding site, a <750 bp (−1490/−20) containing some TA rich repeats and the same 500 bp (−549/−68) genomic region with the binding site mutagenized 5′-AGGAGCATCCT-3′ were separately cloned in a construct containing the Luciferase gene and tested in transcriptional activity assays in P19 cells. When the 500 bp (−549/−68)-Luc construct is cotransfected with pCAG-ArxIRESGFP a reduction in Luciferase expression compared control plasmid pCAG-IRESGFP cotransfection was appreciable (0.67 ± 0.04; n = 3, P < 0.005,); similar reduction was observed when the corepressor TLE1 was cotransfected (0.60 ± 0.07, n = 3); finally, higher reduction was detected when both ARX and TLE1 were cotransfected (0.42 ± 0.06, n = 3). Differently, ARX cotransfected with the 750 bp (−1490/−720)-Luc induced only a slight reduction in the basal transcriptional activity (0.94 ± 0.01, n = 3, P < 0.05,), even in combination with TLE1. When the binding site 5′-ATTAGCATAAT-3′ was mutagenized in 5′-AGGAGCATCCT-3′, no significant reduction in Luciferase expression was appreciable in cotransfection experiments. (I) Immunoprecipitation of E14.5 telencephalic chromatin with anti-ARX, anti-GFP, and IgG only. Primers spanning the 500 bp (−549/−68) genomic region upstream to the Cdkn1c, and Ebf3, Cdkn1b, and Cdkn1a regulative regions were used for PCR analysis of the immunoprecipitated DNA. Enrichment of the PCR products in anti-ARX-immunoprecipitated chromatin sample compared with the anti-GFP and IgG only samples was evaluated.

Article Snippet: Luciferase Reporter Assays P19 cells (10 5 ) were seeded and transfected 24 h after plating, with 400 ng of each of the following constructs in different combinations: pT81 luciferase reporter plasmids, control plasmid pCAG-iresGFP, pCAGArx-iresGFP, pCDNA3-Tle1, and 80 ng of pRL-TK-Renilla luciferase plasmid DNA (Promega) using Lipofectamine PLUS™ Reagent (Roche Diagnostics, Alameda, CA).

Techniques: In Situ, Binding Assay, Sequencing, Clone Assay, Construct, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Cotransfection, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: piggy Bac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential

doi: 10.3390/ijms20194904

Figure Lengend Snippet: In vitro ( A ) and in vivo ( B ) tumorigenic assays. ( A ) Anchorage-dependent growth of MT, MT_E7, and MT_hTERT lines. Cells (including P19 cells) were seeded onto a 2% agar-coated dish, as shown in the left panel. After two weeks, floating cells along with small cell aggregates were observed for all lines ( a – d ). These floating cells were collected and then re-seeded onto a gelatin-coated 60-mm dish, as shown in the left panel. One week after re-seeding, MT, MT_hTRET, and MT_E7 lines failed to attach and spread out onto the surface of a dish ( e – g ), whereas P19 cells attached to the dish and proliferated well ( h ). Bar = 100 μm. ( B ) In vivo tumor formation of MT_E7 cells. Cells (MT_E7 or P19) were injected into an internal area of the pancreas of nude mice under a dissecting microscope, as shown in the left panel. Six weeks after transplantation, generation of distinct solid tumor was discernible in the P19-bearing mice (indicated by arrows enclosed by the dotted lines in ( a )). However, nude mice transplanted with MT_E7 did not show any signs of distinct solid tumor formation (indicated by arrowheads enclosed by the dotted lines in ( b )). When the transplanted region of the pancreas was dissected and inspected, no distinct tumor formation was observed ( c ), although tdTomato-derived red fluorescence was evident ( d ). Bar = 100 μm.

Article Snippet: For inducing solid tumor formation in vivo, MT_E7 (approximately 1 × 10 4 ; expressing tdTomato) or P19 cells (approximately 1 × 10 3 ; used as positive control) were injected into an internal area of the pancreatic parenchyma of nude mice (BALB/cAJcl- nu/nu ; CLEA Japan Ltd., Tokyo, Japan), as described in the left panel of B.

Techniques: In Vitro, In Vivo, Injection, Microscopy, Transplantation Assay, Derivative Assay, Fluorescence

Journal: BMC Pharmacology & Toxicology

Article Title: Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

doi: 10.1186/s40360-017-0151-8

Figure Lengend Snippet: Development of neurons derived from RA-treated P19 and SH-SY5Y cells, and NGF-stimulated PC12 cells up to 10 days in culture. The cells were plated at a density of 500 cells/mm 2 and immunostained against the neuron-specific protein βIII-tubulin. a Representative fluorescence microscopy images of neurons (20 × magnification). b Fluorescence of anti-βIII-tubulin antibodies measured in a microplate reader and expressed as relative fluorescence units (RFU). Data are means ± SEM of 3–4 independent experiments

Article Snippet: P19 mouse embryonal carcinoma cells (cat. no. ECACC 95102107), PC12 rat adrenal pheochromocytoma cells (cat. no. ECACC 88022401) and SH-SY5Y human neuroblastoma cells (cat. no. ECACC 94030304) were obtained from European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Derivative Assay, Fluorescence, Microscopy

Journal: BMC Pharmacology & Toxicology

Article Title: Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

doi: 10.1186/s40360-017-0151-8

Figure Lengend Snippet: Concentration-dependent effects of MeHg, okadaic acid and acrylamide on cell viability and expression of the neuron-specific protein βIII-tubulin in neuronally differentiated P19, PC12 and SH-SY5Y cells. The cells were plated at a density of 500 cells/mm 2 and cultured for 6 days in the differentiation media, followed by exposure to the test compounds for 48 h. Effects of MeHg ( a , b ), okadaic acid ( c , d ) and acrylamide ( e , f ) on the cell viability were assessed by using the calcein-AM assay ( a , c , e ), and the immunofluorescence of βIII-tubulin ( b , d , f ). The data are means ± SEM of n = 6 independent experiments ( n = 3 for okadaic acid in the βIII-tubulin assay; panel d ). The results are expressed as percentage of non-treated cells or cells treated with 0.1% DMSO (used as vehicle). Wells treated with 2% Triton X-100 for 30 min served as controls for maximal cell death. Statistical analysis was performed using repeated measures one-way ANOVA with post hoc Dunnett’s multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001) compared to corresponding controls

Article Snippet: P19 mouse embryonal carcinoma cells (cat. no. ECACC 95102107), PC12 rat adrenal pheochromocytoma cells (cat. no. ECACC 88022401) and SH-SY5Y human neuroblastoma cells (cat. no. ECACC 94030304) were obtained from European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Concentration Assay, Expressing, Cell Culture, Calcein AM Assay, Immunofluorescence

Journal: BMC Pharmacology & Toxicology

Article Title: Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

doi: 10.1186/s40360-017-0151-8

Figure Lengend Snippet: Representative fluorescence microscopy images of neuronally differentiated P19, PC12 and SH-SY5Y cells exposed to 1 μM methylmercury, 10 nM okadaic acid and 1 mM acrylamide. The cells were plated at a density of 500 cells/mm 2 and cultured for 6 days in the differentiation media, followed by exposure to the test compounds for 48 h. The cells were immunolabeled against the neuron-specific protein βIII-tubulin and the fluorescence microscopy images were obtained at 20 × magnification

Article Snippet: P19 mouse embryonal carcinoma cells (cat. no. ECACC 95102107), PC12 rat adrenal pheochromocytoma cells (cat. no. ECACC 88022401) and SH-SY5Y human neuroblastoma cells (cat. no. ECACC 94030304) were obtained from European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Fluorescence, Microscopy, Cell Culture, Immunolabeling

Journal: BMC Pharmacology & Toxicology

Article Title: Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

doi: 10.1186/s40360-017-0151-8

Figure Lengend Snippet: Effects of MeHg, BSO and GSH on the viability of RA-treated P19 cells ( a ) and SH-SY5Y cells ( b ). Cells cultured for 6 days in the differentiation media were pre-treated with 100 μM BSO for 17 h or 1 mM GSH for 1 h followed by exposure to 1 μM MeHg for 24 h. Cell viability was assessed with the PrestoBlue assay that measures cellular metabolic reduction, and extracellular LDH activity assay. Data are means ± SEM of n = 6 independent experiments. For the PrestoBlue assay, data are expressed as percentage of non-treated or 0.1% DMSO vehicle-treated cells. For the LDH assay, the data are presented as percentage of total cell death (cells treated with 2% Triton X-100). Statistical analysis was undertaken using one-way ANOVA with post hoc Bonferroni’s multiple comparisons test: * p < 0.05, ** p < 0.01, and **** p < 0.0001 (the effect of the treatment compared to the corresponding vehicle control, or the effect of the combination of MeHg + GSH or MeHg + BSO compared to MeHg per se ). ND = not determined

Article Snippet: P19 mouse embryonal carcinoma cells (cat. no. ECACC 95102107), PC12 rat adrenal pheochromocytoma cells (cat. no. ECACC 88022401) and SH-SY5Y human neuroblastoma cells (cat. no. ECACC 94030304) were obtained from European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Cell Culture, Prestoblue Assay, Activity Assay, Lactate Dehydrogenase Assay, Control

Journal: BMC Pharmacology & Toxicology

Article Title: Comparison of neurons derived from mouse P19, rat PC12 and human SH-SY5Y cells in the assessment of chemical- and toxin-induced neurotoxicity

doi: 10.1186/s40360-017-0151-8

Figure Lengend Snippet: The effects of MeHg, GSH and BSO on TMRE fluorescence in RA-treated P19 ( a ) and SH-SY5Y cells ( b ). Cells cultured for 6 days in the differentiation media were pre-treated with 100 μM BSO for 17 h or with 1 mM GSH for 1 h followed by exposure to 1 μM MeHg for 24 h. Alterations in mitochondrial membrane potential were measured with the TMRE assay. Data are means ± SEM of n = 6 independent experiments, and expressed as percentage of non-treated or 0.1% DMSO vehicle-treated cells. Statistical analysis was undertaken using one-way ANOVA with post hoc Bonferroni’s multiple comparisons test: * p <0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 (the effect of the treatment compared to the corresponding vehicle control, or the effect of the combination of MeHg + GSH or MeHg + BSO compared to MeHg per se )

Article Snippet: P19 mouse embryonal carcinoma cells (cat. no. ECACC 95102107), PC12 rat adrenal pheochromocytoma cells (cat. no. ECACC 88022401) and SH-SY5Y human neuroblastoma cells (cat. no. ECACC 94030304) were obtained from European Collection of Authenticated Cell Cultures (ECACC) (Porton Down, UK).

Techniques: Fluorescence, Cell Culture, Membrane, Control