Journal:

Article Title: Analysis of Mammalian Cell Cytoplasm with Electrophoresis in Nanometer Inner Diameter Capillaries

doi: 10.1002/elan.200403240

Figure Lengend Snippet: Comparison of the electropherograms obtained using a) 510-fL sample injected from 10-μm diameter PC12 cell, b) 500 μM solution of DA and CAT, and c) sample of physiological saline injected from the cell culture dish. A large neutral peak is almost always observed due to the presence of physiological saline. Subcellular sample and physiological saline injections: 6-s at 3 kV. Standard solution injection: 10-s at 10 kV. All separations were performed using a 25-kV separation potential and 50 mM TES (pH 7.2) separation buffer with 2% 1-propanol.

Article Snippet: Cell Culture PC12 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained as previously described [ 17 ].

Techniques: Comparison, Injection, Saline, Cell Culture

Journal:

Article Title: Analysis of Mammalian Cell Cytoplasm with Electrophoresis in Nanometer Inner Diameter Capillaries

doi: 10.1002/elan.200403240

Figure Lengend Snippet: Electropherograms comparing a) 38-fL sample injected from a 10-μm diameter PC12 cell and b) 1 mM standard solution of DA and CAT. A 5-s injection at 3 kV was employed for both samples. Separation conditions as in Figure 2.

Article Snippet: Cell Culture PC12 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained as previously described [ 17 ].

Techniques: Injection

Journal:

Article Title: Analysis of Mammalian Cell Cytoplasm with Electrophoresis in Nanometer Inner Diameter Capillaries

doi: 10.1002/elan.200403240

Figure Lengend Snippet: Determination of DA in subcellular samples taken from PC12 cells. Separations were performed using a 770 nm i.d. capillary, injection voltages ranging from 2 to 3 kV, injection times ranging from 2 to 5 s, a 25-kV separation potential and a 50 mM TES (pH 7.2) separation buffer with 2% 1-propanol. Cell volumes were crudely estimated using the cell radius and modeling the cell as a sphere. The DA concentrations were calculated using a separation of a standard DA and CAT solution performed on the same capillary and the linear relationship between peak area and amount of analyte.

Article Snippet: Cell Culture PC12 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained as previously described [ 17 ].

Techniques: Injection, Concentration Assay

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: 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

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