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:

Journal: The Journal of Cell Biology

Article Title: Moesin controls cell–cell fusion and osteoclast function

doi: 10.1083/jcb.202409169

Figure Lengend Snippet: (Related to ). (A and B) Depletion of meosin by siRNA in hOC. Western blot analysis of moesin and activated ERM (P-ERM) expression level in hOC after treatment on day 0 with nontargeting siRNA (siCTL) or siRNA targeting moesin (siM), on days 3, 6, and 10 of differentiation. (A) Representative experiment and (B) quantification of moesin (left) and P-ERM (right) expression level normalized to actin. Each circle represents a single donor, n = 7–8, SDs are shown. (C–E) Effect of LPC treatment on hOC fusion and activated ERM expression level (P-ERM). On day 6 of differentiation, hOC were treated with LPC (+LPC, 17 h), or treated and then washed (90 min) (+/− LPC) or no treated (control, CTL). (C and D) Microscopy analysis of hOC fusion. (C) Representative microscopy images: F-actin (phalloidin, white) and nuclei (DAPI, cyan). Scale bar, 50 µm. (D) Quantification of fusion index (left) and area occupied by multinucleated OC (right). Each circle represents a single donor, n =3–4. (E) Western blot analysis of P-ERM expression level in each condition, normalized to actin. Representative blot (left panel) and quantification (right panel). Each circle represents a single donor, n = 4. (F) P-ERM signal by western blot analysis in murine inflammatory osteoclasts (DC-OC) versus control osteoclasts (MN-OC). See material and methods. Representative blot of P-ERM expression level in each condition (upper panel) and quantification of P-ERM expression level, normalized to actin (lower panel). Each circle represents a single mouse, n = 4, SDs are shown. (G–J) Effect of HIV infection on activated ERM expression level (P-ERM) in hOC (G and H) and macrophages (I and J). (G and H) On day 6 of differentiation, hOC were infected with the viral strain NLAD8-VSVG (+HIV-1) or not (CTL) and analyzed 8 days after infection. (G) Quantification of the fusion index (each circle represents a single donor, n = 4) and (H) representative western blot analysis of P-ERM expression level (left), and quantification of P-ERM expression level, normalized to actin (right, each circle represents a single donor, n = 5, SD is shown). (I and J) On day 6 of differentiation, macrophages were infected with the viral strain NLAD8-VSVG (+HIV-1) or not (CTL) and analyzed 7 days after infection. (I) Quantification of the fusion index (each circle represents a single donor, n = 4) and (J) representative western blot analysis of P-ERM expression level (left) and quantification of P-ERM expression level, normalized to actin (right). Each circle represents a single donor, n = 6, SDs are shown. Predicted molecular weights are indicated on western blots. Statistical analyses: (D and E) one-way ANOVA and then Tukey multiple comparison tests. P value is indicated on the graphs, *P ≤ 0.05. Source data are available for this figure: .

Article Snippet: TAT-C3 was added at 48 h, Y27632 at 24 h, NSC23766 and ML141 at 10 h, and calyculin and staurosporine at 10 min. Osteoclast fusion was synchronized with LPC (1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine, #855475; Avanti Polar Lipids) ( ).

Techniques: Western Blot, Expressing, Control, Microscopy, Infection, Comparison

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Representative bright field (BF) and immunofluorescence images of 5% EtOH treatment (vol/vol, 1 and 3 h) induced neurite retraction, and its regeneration after washing EtOH (Washed, 4 and 20 h) in neuronal PC12 cells. Green: neuron-specific β-III tubulin; Blue: DAPI. Scale bar: 50 µm. B , C Quantification results of mean neurite length per cell and the percentage of cells with neurites. *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Immunofluorescence, Control

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Original fluorescence and 3D reconstruction images of Mito-tracker stained mitochondria in neuronal PC12 cells showed mitochondrial fragmentation induced by exposure to EtOH (5%, vol/vol, 1 and 3 h) and subsequent recovery of mitochondrial morphology after washing EtOH (Washed, 4 and 20 h). Scale bar: 20 µm. B Quantification result of the percentage of cells with mitochondrial fragmentation. Data are presented as mean ± SEM. **** P < 0.0001, vs. control group; #### P < 0.0001, vs. EtOH 3 h group. C – E Quantification results of mitochondrial number, mean mitochondrial volume and total mitochondrial volume per cell. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, **** P < 0.0001, vs. control group; ## P < 0.01 , #### P < 0.0001, vs. EtOH 3 h group. F Quantification of mean fluorescence intensity stained with TMRM. Data are presented as mean ± SEM. *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. G ATP content (nmol/mg protein) in neuronal PC12 cells. Data are presented as mean ± SEM. ** P < 0.01, *** P < 0.001, vs. control group; ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. H The relative mtDNA copy number estimated with qPCR measurement. Data are presented as mean ± SEM. EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Fluorescence, Staining, Control

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A Representative images of mitochondrial ultrastructure of neuronal PC12 cells showed EtOH (5%, vol/vol, 1 and 3 h) induced mitochondrial changes such as fragmentation and swelling, and mitochondrial recovery after washing EtOH (Washed, 4 and 20 h). Scale bar: 0.5 µm. B The percentage of mitochondria which appeared as tubes or large and small globes in the sections. C – E Quantification of mitochondria related parameters, including mitochondrial length, perimeter, and area per mitochondrion. At least 300 mitochondria were quantified in each group. Data are presented as mean ± SD. *** P < 0.001, vs. control group; ### P < 0.001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Control

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A – D Protein expression of LC3-I/II, p62, Parkin, PINK1 in neuronal PC12 cells detected by western blot. GAPDH was used as a loading control. E Immunofluorescence images represent LC3 dots (green) and nuclei (blue) in neuronal PC12 cells. Scale bar: 10 µm. F , G Quantification of the number of LC3 dots per cell and the percentage of cells with LC3 dots. Data are presented as mean ± SEM. **** P < 0.0001, vs. control group. H Electron microscopy showed autophagy induced by EtOH (5%, vol/vol, 1 and 3 h) in neuronal PC12 cells. The arrows point to the autophagosome (double membrane) and autolysosome (single membrane). Scale bar: 1 µm. I , J Quantification of the number of LC3 dots per cell. Data are presented as mean ± SEM. *** P < 0.001 , **** P < 0.0001, vs. control group. #### P < 0.000, vs. EtOH 1 h group. ^^ P < 0.01, ^^^^ P < 0.0001, vs. EtOH 3 h group. K – N Quantification results of mean neurite length per cell, the percentage of cell with neurite, the percentage of cells with fragmented mitochondria, and TMRM mean fluorescence intensity, which show that the autophagy inhibitor 3-MA had no inhibitory effect on the cell recovery from EtOH induced damage. EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Electron Microscopy, Membrane, Fluorescence, Cell Recovery

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A – D mRNA expression of Drp1, OPA1, MFN1, and MFN2 measured by qPCR in neuronal PC12 cells. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 , vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. EtOH 3 h group. E – K Protein expression of Drp1, p-Drp1 (Ser616), p-Drp1 (Ser637), OPA1, MFN1, and MFN2 in neuronal PC12 cells detected by Western blot. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05, ## P < 0.01 , ### P < 0.001, vs. EtOH 3 h group. L Immunofluorescence images showed that Drp1 signals were diffused in the cytosol before EtOH treatment. After applying EtOH for 3 h, the signals became more punctate and appeared to be translocated to the mitochondria. After removing EtOH by washing, the Drp1 signals were again diffused throughout the cytosol. Mitochondria and Drp1 were stained with TOM20 antibody (red) and Drp1 antibody (green) separately. Scale bar: 10 µm. EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Expressing, Control, Western Blot, Immunofluorescence, Staining

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A – C mRNA expression of PGC-1α, AMPK-1α, SIRT1 in neuronal PC12 cells measured by qPCR. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, vs. control group; ## P < 0.01, #### P < 0.0001, vs. EtOH 3 h group. D – H Protein expression of PGC-1α, AMPK-1α, p-AMPK-1α, and SIRT1 in neuronal PC12 cells detected by western blot. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. control grou p ; # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, vs. EtOH 3 h group. EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Expressing, Control, Western Blot

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: Bright field images of neuronal PC12 cells with or without EtOH treatment for 3 h, and after washing EtOH then further culturing cells in fresh medium for another 4 or 20 h with or without different concentration of SR-18292 (50, 75, 100 µM). EtOH ethanol.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Concentration Assay

Journal: Cell Death Discovery

Article Title: PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration

doi: 10.1038/s41420-024-01953-0

Figure Lengend Snippet: A – C Quantification of cell viability, the percentage of cells with neurites, and the mean neurite length per cell in neuronal PC12 cells treated alone with different concentration of SR-18292 (50, 75, 100 µM) for 4 and 20 h. Data are presented as mean ± SEM. * P < 0.05, vs. control group. D – F Quantification of cell viability, the percentage of cells with neurites, and the mean neurite length per cell in neuronal PC12 cells with corresponding treatment. Data are presented as mean ± SEM. **** P < 0.0001, vs. control group; ## P < 0.01, #### P < 0.0001, vs. EtOH 3 h group; ^^^^ P < 0.0001, vs. Washed 4 h group; !!!! P < 0.0001, vs. Washed 20 h group. G , H Quantification of the percentage of cells with fragmented mitochondria and the TMRM mean fluorescence intensity in neuronal PC12 cells treated alone with different concentrations of SR-18292 (50, 75, 100 µM) for 4 and 20 h. I , J Quantification of cell viability, the percentage of cells with neurites, and the mean neurite length per cell in neuronal PC12 cells with corresponding treatment. Data are presented as mean ± SEM. **** P < 0.0001, vs. control group; #### P < 0.0001, vs. EtOH 3 h group; ^^^^ P < 0.0001, vs. Washed 4 h group; !!!! P < 0.0001, vs. Washed 20 h group.

Article Snippet: Pheochromocytoma line 12 (PC12) cells were obtained from Leibniz Institute DSMZ (ACC 159) and cultured as described before, with minor modifications [ ].

Techniques: Concentration Assay, Control, Fluorescence