Journal: The Journal of Cell Biology

Article Title: The novel gene asb11 : a regulator of the size of the neural progenitor compartment

doi: 10.1083/jcb.200601081

Figure Lengend Snippet: Effect of d-Asb11 on differentiation in PC12 or Nt2-D1 cells. (A) Proliferation of PC12 cells upon NGF stimulation. Relative mitochondrial activity was measured (MTT assay), and PCNA expression levels were determined. The experiments were performed in duplicate, and the values shown are the means and SEM (error bars) of 11 different samples. (B) Neurite length was measured in at least 10 different fields of cells for each condition. **, P < 0.01. (C) Fluorescent double labeling of PC12 cells transfected with MT or MT–d-Asb11. d-Asb11 inhibited neurofilament (NF) expression and allowed continued cell proliferation as compared with MT-transfected cells. (D) The protein expression profile of GAP-43 and neurofilament of whole cell lysates in PC12 cells after NGF stimulation. (E) Expression of neurofilament, PH3 (Ser10), and actin in Nt2-D1 cells after RA addition. Bars, 200 μm.

Article Snippet: Blots were incubated overnight at 4°C in TBS-T (50 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) containing 10% blocking buffer and 1:1,000 PCNA (Sigma-Aldrich), GAP-43 (Sigma-Aldrich), neurofilament (for PC12; 2H3; Developmental Studies Hybridoma Bank), neurofilament-M (for Nt2-D1; Cell Signaling), or β-actin (SC-1615; Santa Cruz Biotechnology, Inc.).

Techniques: Activity Assay, MTT Assay, Expressing, Labeling, Transfection

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: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: Axonal p75 is retrogradely transported in a ligand- and dynein-dependent manner. ( a ) Illustration of the design of the experiment used to study retrograde transport and signaling in sympathetic neurons. ( b ) Kymographs of axonal p75 movement in response to BDNF. Compartmentalized sympathetic neuron cultures were treated with BDNF (200 ng/mL) and MC192-QD, which labeled the extracellular domain of p75, for 4 hours at 37 °C in the axon compartment before imaging. Upper panels show kymographs of retrograde p75 movement in the absence (−) and presence (+) of BDNF in the axon compartment. ( c ) The graph indicates the proportion of vesicles without (black) or with (gray) movement when axon chambers were treated without (−) or with (+) BDNF. Data were obtained from videos captured in three independent experiments. ( d ) Histogram showing the distribution of velocities measured from 105 moving vesicles. The mean velocity of p75 vesicles undergoing retrograde transport in the presence of BDNF was approximately 1.5 µm/second. ( e ) Illustration of the design of the experiment used to study the retrograde transport of p75 and its dependence on dynein. ( f ) Visualization of axonally labeled p75 in the cell bodies of compartmentalized sympathetic neurons. The axons of compartmentalized neuronal cultures were treated with BDNF and MC192-Alexa Fluor 594 (to label p75, red) and microspheres labeled with Alexa Fluor 488 (to label compartmentalized neurons, green) for 16 hours at 37 °C in the absence (control) or presence of the dynein inhibitor EHNA (erythro-9-(2-hydroxy-3-nonyl). Scale bar, 10 μm. ( g ) Levels of axonally labeled p75 in the absence (control) or presence of EHNA. Data from 120 cells in four different compartmentalized chambers were quantified. Microspheres labeled with Alexa Fluor 488 were added 24 hours before the BDNF treatment and maintained in the culture throughout the experiment. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Labeling, Imaging, Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: Rab5 activity is required for p75 retrograde transport and axonal BDNF-induced death signaling in sympathetic neurons. ( a ) Illustration of the design of the experiment used to study Rab5-dependent p75 retrograde transport and signaling in sympathetic neurons. Quantification ( b ) and visualization ( c ) of axonally labeled p75 in the cell bodies of compartmentalized sympathetic neurons. ( b ) Levels of axonally labeled p75 in the absence (control, GFP) or presence of a dominant negative Rab5 mutant (Rab5DN-GFP). Sixty cells from five different compartmentalized chambers were quantified. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001. ( c ) After 5 days in culture, sympathetic neurons were transduced with the adenovirus driving the expression of GFP (green) or a dominant negative mutant of Rab5 fused to GFP (Rab5DN-GFP, green). Twenty hours after the infection, the axon compartment was treated with BDNF, MC192-Alexa Fluor 594 (to label p75, red) and the B subunit of the cholera toxin conjugated to Alexa Fluor 647 (B-CTX, cyan) for 16 hours at 37 °C to label compartmentalized neurons. Scale bar, 10 μm. Only cells that were retrogradely labeled with B-CTX-Alexa Fluor 647 were used to quantify the retrograde transport of p75. The white arrow indicates a Rab5DN-GFP labelled neuron with reduced levels of p75. The arrowhead indicates a neuron expressing lower labels of Rab5DN-GFP when compared to the neuron labelled with a white arrow. Consistently, this neuron shows increased levels of p75. The asterisk indicates a neuron that does not express Rab5DN-GFP. This neuron expresses greater levels of p75 compared to neurons labelled with Rab5DN-GFP. ( d ) Illustration of the design of the experiment used to study Rab5-dependent retrograde signaling of p75 and quantify axonal BDNF-induced death signaling in sympathetic neurons. Sixty cells from seven different compartmentalized chambers were quantified at each time point. Statistically significant differences were analyzed using two-way ANOVA and Tukey’s multiple comparison test. ****p < 0,0001. Only cells that were retrogradely labeled with B-CTX-Alexa Fluor 647 were used to quantify apoptosis. ( e-f ) Quantification ( e ) and visualization ( f ) of apoptosis of neuronal cell bodies from compartmentalized sympathetic neuronal cultures expressing GFP and Rab5DN-GFP (in green). Axons were treated with B-CTX-Alexa Fluor 647 (red) to label compartmentalized neurons. Condensed or fragmented nuclei were labeled with Hoechst (blue). The cell enclosed in a white square was magnified to enable the better visualization of the healthy or condensed nucleus labeled with an asterisk. The white arrow indicates a healthy nucleus that is not labeled with B-CTX-Alexa Fluor 647. Scale bar, 10 μm.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Activity Assay, Labeling, Dominant Negative Mutation, Mutagenesis, Two Tailed Test, MANN-WHITNEY, Transduction, Expressing, Infection

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: JNK promotes p75 internalization in the axons and cell bodies of sympathetic neurons. ( a ) and ( b ) Visualization of axons of compartmentalized cultures of sympathetic neurons treated with BDNF and MC192-pHRodo in the absence (vehicle) or presence of dynasore for 3–4 hours at 37 °C ( a ), or the absence (vehicle) or presence of SP600125 for 3–4 hours at 37 °C ( b ). The intensity of the pHRodo fluorophore is brighter at an acidic pH, indicating p75 internalization. An inhibitor of p75 internalization, dynasore, reduced the fluorescence associated with p75 internalization. Similar to the effect of dynasore, the presence of SP600125 reduces the fluorescence compared to vehicle conditions. Scale bar, 4,5 µm. Right panels show the quantification of the total fluorescence associated with 366 (vehicle for dynasore), 406 (dynasore), 268 (vehicle for SP600125), 227 (SP600125) axonal segments (9 μm long) from three independent compartmentalized neuronal cultures. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001. ( c ) and ( d ) Confocal microscopy images of sympathetic neurons treated with BDNF (150 µg/mL) and MC192-Alexa Fluor 594 (3 μg/mL, green, to label p75) for 4 hours at 37 °C in the absence (vehicle and TAT-TI control peptide) or presence of the JNK inhibitors, SP600125 (10 µM) ( c ) or TAT-TI-JIP 153–163 peptide (1 μM) ( d ). Scale bar, 10 μm. Right panels show the levels of internalized p75 after different treatments (relative fluorescence normalized to cell surface p75). Sixty-five cells from three independent experiments were quantified. Statistically significant differences were analyzed using a two-tailed Mann-Whitney test. ****p < 0,0001.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Fluorescence, Two Tailed Test, MANN-WHITNEY, Confocal Microscopy

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase (JNK)-dependent internalization and Rab5-dependent endocytic sorting mediate long-distance retrograde neuronal death induced by axonal BDNF-p75 signaling

doi: 10.1038/s41598-019-42420-6

Figure Lengend Snippet: JNK is required for the efficient retrograde transport of p75-positive endosomes. ( a ) Schematic of the methodology; the axonal compartment of sympathetic neurons was treated with MC192 conjugated with QD605 and BDNF for 4 hours. Thirty minutes prior to the time-lapse recording, the medium was changed in both compartments to remove the MC192-QDot. Then, the cell body compartment was treated with SP600125 or ciliobrevin D (20 μM) for 30 minutes. Finally, time-lapse images of the cell body compartment were captured at a site proximal to the microgrooves to evaluate the local effects of the inhibitors on the axonal transport of previously endocytosed MC192-QDot. ( b ) Representative kymograph of the retrograde transport of MC192-QDot in axons treated with vehicle, SP600125 or ciliobrevin D. Scale bar, 20 μm. ( c ) Quantification of the number of mobile (>5 μm traveled) and static (<5 μm traveled) particles in axons. Twelve videos from 4 chambers per condition in 2 independent experiments were quantified and analyzed using two-way ANOVA and Tukey’s multiple comparison test. **p < 0.005 and ***p < 0.001. ( d ) Quantification of endosomal flux, as evidenced by the number of dots observed during 30 seconds in 50 μm of axon multiplied by the average velocity of the particles during the time-lapse in each video. Three videos per chamber under each condition were quantified in 2 independent experiments. Student’s t-test: *p < 0.05. ( e ) Graph of the average velocity of the particles under each condition. Fifty-eight to 106 particles per condition were quantified in 2 independent experiment. Statistical significance was determined using Student’s t-test. p = 0.5868. ( f ) Graph indicating the ratio of the average distance traveled by the moving particles under each condition. Fifty-eight to 106 particles per condition were quantified in 2 independent experiments. Statistical significance was determined using Student’s t-test. p = 0.3249. ( g ) In sympathetic neurons axons, BDNF bound to p75 is internalized in a JNK-dependent manner to Rab5 positive endosomes and then is sorted to apoptosis-signaling endosomes for dynein mediated retrograde transport.

Article Snippet: NGF, BDNF, anti-BDNF and the monoclonal MC192 anti-p75 antibody were purchased from Alomone Labs (Jerusalem, Israel).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Pro-nerve Growth Factor Induces Autocrine Stimulation of Breast Cancer Cell Invasion through Tropomyosin-related Kinase A (TrkA) and Sortilin Protein *

doi: 10.1074/jbc.M110.211714

Figure Lengend Snippet: Effect of proNGF on invasion of breast cancer cells and associated signaling pathways. A, cell invasion assay on MDA-MB-231 transfected with the siRNA against proNGF (siproNGF), p75NTR (sip75), sortilin (siSORT), TrkA (siTrkA), control siRNA (Control), or TrkA kinase-dead versus TrkA wild type, treated or not with 0.5 nm recombinant human non-cleavable proNGF (N.C. proNGF) and/or 10 nm K252a (Trk inhibitor), 15 μm LY294002 (PI3 kinase inhibitor), 50 nm SKI-1 (Src inhibitor), or 10 μm PD98059 (MAP kinase inhibitor). Untreated siGFP-transfected control cells represent the control 100% of invasion (white bar). The efficiency of siRNA treatments was assessed by Western blotting. B, proNGF-induced cell signaling in breast cancer cells. MDA-MB-231 cells were stimulated by 0.5 nm non-cleavable proNGF for the indicated times. C, the efficacy and specificity of pharmacological inhibitors used in cell invasion assays were tested in Western blotting. D, cell invasion assay on MDA-MB-231 cells transfected with mutated forms of TrkA. Tyr-490, Tyr-695, Tyr-751, and Tyr-785 were mutated, and response to proNGF was tested. E, neurotensin effect on proNGF-induced signaling and breast cancer cell invasion. For Western blotting and cell invasion assay, the experimental conditions were identical to what was described in A and B. For the statistics in A, D, and E, error bars represent S.D. *, p < 0.001 for proNGF stimulation versus no stimulation; §, p < 0.001 for experimental versus control under proNGF stimulation; ¶, p < 0.001 for experimental versus control with no proNGF stimulation.

Article Snippet: The decrease in targeted protein level was assessed by immunoblotting with anti-proNGF (AB9040, Millipore), anti-p75 NTR (clone D8A8, Cell Signaling Technology), anti-TrkA (Sc-118, Santa Cruz Biotechnology), and anti-sortilin (612101, BD Biosciences or ANT-009 Alomone Labs, for detection of rat sortilin in PC12 cells).

Techniques: Invasion Assay, Transfection, Recombinant, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Pro-nerve Growth Factor Induces Autocrine Stimulation of Breast Cancer Cell Invasion through Tropomyosin-related Kinase A (TrkA) and Sortilin Protein *

doi: 10.1074/jbc.M110.211714

Figure Lengend Snippet: ProNGF versus NGF effect on TrkA-mediated signaling and stimulation of breast cancer cell invasion. A, ProNGF does not require cleavage into NGF to stimulate TrkA activation and breast cancer cell invasion. The furin inhibitor I was tested at the indicated concentration on both MDA-MB-231 cell invasion and TrkA signaling activation as described under “Experimental Procedures.” N.C. proNGF, non-cleavable proNGF. B, dose effect of proNGF and NGF on breast cancer cell invasion. The indicated concentrations were tested for 20 h. C, cell invasion assay on MDA-MB-231 transfected with the siRNA against p75NTR (sip75), sortilin (siSORT), TrkA (siTrkA), control siRNA (Control), or TrkA kinase-dead versus TrkA wild type, treated or not with 0.5 nm recombinant human NGF and/or 10 nm K252a (Trk inhibitor), 15 μm LY294002 (PI3 kinase inhibitor), 50 nm SKI-1 (Src inhibitor), or 10 μm PD98059 (MAP kinase inhibitor). Untreated siGFP-transfected cells represent the control 100% of invasion (white bar). D, NGF-induced cell signaling in breast cancer cells. MDA-MB-231 cells were stimulated with 16 nm NGF for the indicated times. For statistics in A, B, and C, error bars represent S.D. *, p < 0.001 for proNGF stimulation versus no stimulation; p < 0.001 for experimental versus control under proNGF stimulation; §, p < 0.001 for experimental versus control under proNGF stimulation; ¶, p < 0.001 for experimental versus control with no proNGF stimulation. ‡, p < 0.001 for proNGF versus NGF at the same concentration. E, side-by-side comparison of signaling intensity, obtained on the same blot, for proNGF and mature NGF. Concentrations for which a stimulation of breast cancer cell invasion was obtained, i.e. 0.5 nm proNGF and 16 nm mature NGF, were used.

Article Snippet: The decrease in targeted protein level was assessed by immunoblotting with anti-proNGF (AB9040, Millipore), anti-p75 NTR (clone D8A8, Cell Signaling Technology), anti-TrkA (Sc-118, Santa Cruz Biotechnology), and anti-sortilin (612101, BD Biosciences or ANT-009 Alomone Labs, for detection of rat sortilin in PC12 cells).

Techniques: Activation Assay, Concentration Assay, Invasion Assay, Transfection, Recombinant

Journal: Nature Communications

Article Title: Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity

doi: 10.1038/s41467-023-43716-y

Figure Lengend Snippet: a Dependency map of Rb from the Cancer Dependency Map Project . b Percentage of proliferating cells in wild-type and tKO MCF-10A cells treated with either DMSO or palbociclib (1 µM). Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05; ** p ≤ 0.001). c Heatmap of single-cell traces for CDK4/6 and CDK2 activities and Cdt1 degron levels in tKO MCF-10A cells treated with palbociclib (1 µM). During 25‒48 h after drug treatment, cells activating CDK2 (>1) for over 2 h were classified as proliferating cells. d Percentage of S-phase cells in tKO MCF-10A cells without and with expression of a DHFR-p27 construct. Data are shown as mean ± SD ( n = 4 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05). e Average traces of CDK2 activity and p53 and p21 levels in MCF-7 cells treated with palbociclib (1 µM). Data are shown as mean ± 95% CI (proliferation: n = 447 cells; quiescence: n = 3582 cells). f , g Accumulative percentage of S-phase cells in tKO ( f ) or wild-type ( g ) MCF-10A cells. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the one-way ANOVA test (* p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001). h Percentage of S-phase cells in palbociclib-resistant MCF-10A cells exposed to different concentrations of mitogens and NCS as indicated for 24 h. Data are shown as mean ± SD ( n = 4 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05).

Article Snippet: Anti-p21 (#sc-271610; 1:300 for immunoblotting in PC-12 and OP-9 cells) was ordered from Santa Cruz Biotechnology.

Techniques: Two Tailed Test, Expressing, Construct, Activity Assay

Journal: Nature Communications

Article Title: Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity

doi: 10.1038/s41467-023-43716-y

Figure Lengend Snippet: a Percentage of S-phase cells in PLB-985, PC-12, and OP-9 cells after differentiation for the indicated time. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (PC-12 cells) or one-way ANOVA test (PLB-985 and OP-9 cells) (* p ≤ 0.05; ** p ≤ 0.001; *** p ≤ 0.0001). b Percentage of neurite bearing PC-12 cells before and after one-day differentiation. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05). c Percentage of high-PPARγ expressing OP-9 cells. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the one-way ANOVA test (* p ≤ 0.05; ** p ≤ 0.001). d Immunoblot showing Rb, c-Myc, p21, p27, and GAPDH expression before and after differentiation. FPR1, SYN1, and PPARγ are cell differentiation markers. e Percentage of S-phase OP-9 cells expressing a doxycycline-inducible c-Myc construct. After 6 days of differentiation, cells were treated with palbociclib (1 µM) and EdU (10 µM) + DMSO or doxycycline (5 µM) for 72 h. Data are shown as mean ± SD ( n = 3 biological replicates). Asterisks indicate significant differences in the two-tailed unpaired t -test (* p ≤ 0.05). f Schematic diagram illustrating CDK4/6-independent cell-cycle entry by multiple steps: (1) reduction in Rb-protein levels, (2) c-Myc-mediated amplification of E2F activity, and (3) inhibition of CDK2 activity by Cip/Kip.

Article Snippet: Anti-p21 (#sc-271610; 1:300 for immunoblotting in PC-12 and OP-9 cells) was ordered from Santa Cruz Biotechnology.

Techniques: Two Tailed Test, Expressing, Western Blot, Cell Differentiation, Construct, Amplification, Activity Assay, Inhibition

Journal: Frontiers in Physiology

Article Title: Sedentary Conditions Promote Subregionally Specific Changes in Brain-Derived Neurotrophic Factor in the Rostral Ventrolateral Medulla

doi: 10.3389/fphys.2021.756542

Figure Lengend Snippet: Primary antibodies.

Article Snippet: p75NTR , Peptide CEEIPGRWITRSTPPE, corresponding to amino acids 188–203 of human p75NTR (extracellular domain) , Alomone Labs, ANT-007, rabbit polyclonal , AB_2039968 , 1:200.

Techniques: Recombinant

Journal: Frontiers in Physiology

Article Title: Sedentary Conditions Promote Subregionally Specific Changes in Brain-Derived Neurotrophic Factor in the Rostral Ventrolateral Medulla

doi: 10.3389/fphys.2021.756542

Figure Lengend Snippet: Expression of Glycosylated (Glyco-p75) and non-Glycosylated (nonGlyco-p75) forms of the p75NTR receptor in the RVLM/RVLM RE following 12weeks of sedentary vs. physically active conditions. (A) Representative Western blot of Glyco-p75 (arrow ~75kDa), nonGlyco-p75 (arrow ~50kDa), and GAPDH expression at different rostrocaudal levels of the RVLM and the RVLM RE . (B) Group data from sedentary (black bars) vs. physically active (white bars) conditions ( n =6 ea) demonstrate no significant overall difference in the expression of Glyco-p75 in sedentary compared to active animals [ F (1, 30)=1.150, p =0.309, main effect of group] and there was also no overall significant effect of rostrocaudal distribution [ F (3,30)=1.877, p =0.155; main effect]. The interaction between experimental groups and rostrocaudal levels did not reach a significance [ F (3,30)=2.029, p =0.131], which precluded further post hoc testing. (C) Group data from sedentary vs. physically active conditions demonstrate a significant interaction term [ F (3,30)=3.384, p =0.031] and revealed a significantly lower expression of nonGlyco-p75 in both RVLM subregions ( ** p =0.004 for FN-480 and p =0.006 for FN-240) and in the FN+240 subregion of the RVLM RE ( ** , p =0.001) of sedentary rats. Sedentary rats showed significantly higher expression of nonGlyco-p75 in the FN+480 of the RVLM RE compared to the FN+240 (##, p =0.012) and both RVLM subregions (##, p =0.004 for FN-240 and p <0.001 for FN-480). Physically active rats exhibited significantly higher expression of nonGlyco-p75 in the FN+240 subregion of RVLM RE compared with the FN-480 of RVLM (##, p =0.006). See for results of all rostrocaudal comparisons within groups. (D) Group data showing that the Glyco-p75/nonGlyco-p75 ratio was overall significantly higher in sedentary rats vs. physically active [*, F (1,30)=20.829, p =0.001, main effect] and that there was an overall significant main effect of rostrocaudal distribution [#, F (3,30)=7.205, p <0.001, main effect]. Simple main effect testing revealed a significantly higher Glyco-p75/nonGlyco-p75 ratio in the most caudal FN-480 subregion of the RVLM compared with two subregions (FN+240 and FN+480) of the RVLM RE ( p =0.009 and p <0.001, respectively, see for all simple main effect comparisons). The interaction between main effects did not reach a significance [ F (3,30)=2.895, p =0.051] which precluded further post hoc testing. Data in (C,D) were log10 transformed in order to achieve normal distribution prior to running two-way mixed ANOVAs.

Article Snippet: p75NTR , Peptide CEEIPGRWITRSTPPE, corresponding to amino acids 188–203 of human p75NTR (extracellular domain) , Alomone Labs, ANT-007, rabbit polyclonal , AB_2039968 , 1:200.

Techniques: Expressing, Western Blot, Transformation Assay