atcc crl 1721 1 pc12 strain  (ATCC)

Journal: Cell Death Discovery

Article Title: Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease

doi: 10.1038/cddiscovery.2015.27

Figure Lengend Snippet: Effects of MGV-1 (and NGF and glutamate, as well as the various combinations of these three compounds) on PC12 cells regarding viability and differentiation. ( a ) As opposed to U118MG cells, together with 35 mM of glutamate, MGV-1 (10, 25, 50, 100 μ M as indicated at the x axis) induces enhanced cell death of PC12 cells, as compared with application of 35 mM of glutamate by itself. At 100 μ M, MGV-1 by itself can induce cell death of PC12 cells. ( b ) Separately and synergistically, MGV-1 (50 μ M) and glutamate (35 mM) induce collapse of the ΔΨm in PC12 cells. ( c ) Representative examples of different PC12 cell strains (see Materials and methods section): strain #1 (only flat, attached polygonal cells), strain #2 (round cells and polygonal cells), and strain #3 (floating clusters of round cells and a restricted number polygonal cells). MGV-1 can induce differentiation of various strains of PC12 cells. The top row of this c presents undifferentiated cells, and the bottom row of the c presents typical examples of differentiated cells of the three strains. As the morphologies of the undifferentiated cells of the three strains are different, the morphologies of these cell strains differentiated by our applications are also distinct. Neurite sprouting from strain #1 makes the cells appear star shaped (here differentiated by MGV-1+glutamate). Strain #2 gives rise to extended thin neurites (here differentiated by MGV-1+glutamate). Strain (3) gives rise to very long thin neurites (here differentiated by MGV-1+NGF). ( d ) Table: MGV-1, NGF, and glutamate, separately and combined, can induce neurodifferentiation of strains of PC12 cells presenting both spherical and polygonal cells (strains 2 and 3). The lower the total number of cells, the more the cells are differentiated (differentiated cells do not proliferate). The higher the number of cells with neurite (the hallmark of differentiation), the more the cells are differentiated. The longer the average neurite length, the more the cells are differentiated. The strain presenting only polygonal cells (strain #1), can be differentiated by MGV-1 by itself, whereas NGF and glutamate by themselves do not have this effect on cells of strain #1 (as demonstrated as 0 cells presenting a neurite, that is, neurites of 0 length). The shading in the Table for all treatments is according to rank each time in one column (see ‘key’ giving the shading for each of the 8 ranks). The murkiest shading for each parameter (total number of cells, cells with neurite, average neurite length) presents the lowest indication of differentiation (typically the control), the brightest shading indicates the most effective differentiation. Summing up the ranks of each row (presented in the most right-hand column) it was found, looking at the individual treatments of Glu, NGF, and MGV-1, that: MGV-1 works better than NGF works better than glutamate. Interestingly, measuring the percentages of differentiated cells as part of the total cell population remaining per plate, gives the exact same rank order of effectiveness. Regarding combinations of molecules: (MGV-1+Glu+NGF) works better than (MGV-1+Glu) works better than (MGV-1+NGF) works better than (NGF+Glu). Looking at each cell type regarding capacity of differentiation (comparing each parameter for each cell strain and each treatment), the rank order of capacity to differentiate is: Strain #3>Strain #2>Strain #1. Statistical significance following one-way ANOVA and post hoc Mann–Whitney: * P <0.05; ** P <0.01; *** P <0.001. The scale bars in c are 100 μ M.

Article Snippet: This latter strain is similar in appearance to the ATCC CRL-1721.1 PC12 strain (own observations).

Techniques: Control, MANN-WHITNEY

Journal: Cell Death Discovery

Article Title: Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease

doi: 10.1038/cddiscovery.2015.27

Figure Lengend Snippet: Localization of tubulin 3 β ( a – e ) and NeuN ( f – j ) in cell bodies and neurites of differentiated PC12 (strain #3). This figure shows that our different protocols not only result in extensive sprouting and outgrowth of neurites of PC12 cells in culture (as shown in ), but also labeling of these cells with the neuronal markers tubulin 3 β (magenta in a – e ) and NeuN (yellow in f – j ). The cell nuclei are labeled with DAPI (cyan in a – j ). (a ) Tubulin 3 β labeling can be detected first of all in the cell bodies of the undifferentiated vehicle control PC12 cells (control). Inducing differentiation with MGV-1 ( b ), MGV-1 plus glutamate ( c ), NGF ( d ), as well as MGV-1 plus NGF plus glutamate ( e ) enhanced tubulin 3 β labeling not only of the cell body but also intensely of neurites. ( f ) NeuN expression is indicated with yellow fluorescent immunocytochemical labeling of the cell bodies, both in the nuclei and the cytoplasm of undifferentiated cells (control). Nuclei and cytoplasm both are typical locations for NeuN. NeuN labeling can also appear in the neurites of cells differentiated with MGV-1 ( g ), MGV-1 plus glutamate ( h ), NGF ( i ), as well as MGV-1 plus NGF plus glutamate ( j ). NeuN labeling can also appear in the neurites. In undifferentiated as well as differentiated cells doubly labeled for DAPI and NeuN, the cell nuclei can appear whitish, indicating the presence of NeuN in the cell nuclei. The same is true Tubulin for cells doubly labeled for DAPI and tubulin. The scale bars in are 100 μ M.

Article Snippet: This latter strain is similar in appearance to the ATCC CRL-1721.1 PC12 strain (own observations).

Techniques: Labeling, Control, Expressing

Journal: Cell Death Discovery

Article Title: Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease

doi: 10.1038/cddiscovery.2015.27

Figure Lengend Snippet: Neuroimmunochemical signs of differentiation of PC12 cells by our different exposure protocols, applied to Strain #1 ( a – c ) and Strain #3 ( d and e ) in comparison with their vehicle controls. ( a ) A bar graph showing protein content indicative of cell size of strain #1 differentiated by glutamate, MGV-1, and MGV-1+glutamate. MGV-1+glutamate enhances protein levels in these cells sixfold. ( b ) A bar graph of relative tubulin 3 β expression in strain #1 cells differentiated by three different treatments (glutamate, MGV-1, and MGV-1+glutamate), compared with the vehicle control (undifferentiated cells). MGV-1+glutamate significantly enhances tubulin 3 β expression in these cells. ( c ) Representative western blot assay of the effects on the expression levels of tubulin 3 β of figures ( b ). ( d ) A bar graph showing significantly enhanced NeuN expression in cells of strain #3 differentiated by MGV-1+glutamate and by MGV-1+NGF+glutamate, compared with the vehicle control (undifferentiated cells). The other treatments shown (glutamate, MGV-1, NGF, NGF+MGV-1, NGF+glutamate) do not enhance NeuN expression significantly. ( e ) A representative western blot assay of NeuN expression in cells of strain #3 differentiated by our various protocols of . In ( b ) and ( d ), protein expression is given in arbitrary units (× 10 7 ) as provided the ImageQuant LAS 4010 densitometer. Data presented as means±S.E.M. For 5 a and 5 b n =4, for 5 d n =6. In all cases, statistical analysis by the Friedman ANOVA test, and Dunn's multiple comparison test as the post hoc . * P <0.05, ** P <0.01, *** P <0.001 as compared with vehicle control (control). Control, vehicle only; glu, glutamate; M, molecular weight (50 kDa MW) markers for the western blots.

Article Snippet: This latter strain is similar in appearance to the ATCC CRL-1721.1 PC12 strain (own observations).

Techniques: Comparison, Expressing, Control, Western Blot, Molecular Weight

Journal: Cell Death Discovery

Article Title: Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease

doi: 10.1038/cddiscovery.2015.27

Figure Lengend Snippet: Summary of the effects of MGV-1 in cell cultures of U118MG and PC12 cells. Glutamate (35 mM) induces cell death of U118MG cells as well PC12 cells (skulls in top boxes). MGV-1 protects U118MG cells from glutamate-induced cell death (crossed out skulls in left-hand bottom box). In contrast, MGV-1 together with glutamate induces pronounced neuronal differentiation of PC12 cells (image of a mature neuron in most right-hand bottom box). As shown in , MGV-1 also enhances cell death induction of glutamate of PC12 cells. Thus, MGV-1 appears to be able to regulate astrocytic integrity, neuronal differentiation, and weeding out of non-differentiating progenitor cells. ,

Article Snippet: This latter strain is similar in appearance to the ATCC CRL-1721.1 PC12 strain (own observations).

Techniques: