Journal: Frontiers in Molecular Neuroscience

Article Title: Amyloid β1-42 (Aβ1-42) Induces the CDK2-Mediated Phosphorylation of Tau through the Activation of the mTORC1 Signaling Pathway While Promoting Neuronal Cell Death

doi: 10.3389/fnmol.2017.00229

Figure Lengend Snippet: Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.

Article Snippet: The antibodies of hypoxia inducible factor (HIF1α; NB100-105), p70S6K1 (NB600-1049), LC3 (NB100-2220) and p62 (NBP1-48320) were obtained from Novus Biologicals (Littleton, CO, USA) and the HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Incubation, Phospho-proteomics, Immunoprecipitation, Confocal Microscopy, MTT Assay, Trypan Blue Exclusion Assay, Control

Journal: BMC Cancer

Article Title: MARCH1 negatively regulates TBK1-mTOR signaling pathway by ubiquitinating TBK1

doi: 10.1186/s12885-024-12667-y

Figure Lengend Snippet: STING mediates the indirect interaction between MARCH1 and TBK1. A MARCH1 did not interact with TBK1. HEK293T cells expressing the indicated proteins were lysed with NETN lysis buffer and then immunoprecipitated with flag beads to detect these proteins. B MARCH1 interacted with STING. HEK293T cells transfected with the indicated plasmids were lysed with NETN lysis buffer and then immunoprecipitated with flag beads to detect the indicated proteins. 10 μM MG132 was added to the cells for 12 h prior to them being lysed. C MARCH1 interacted with the TBK1 in the presence of STING. HEK293T cells expressing the indicated proteins were treated as described in B . D The interaction between TBK1 and mTOR was enhanced in response to insulin stimulation. HEK293T cells expressing the indicated proteins were starved of serum for 16 h and re-stimulated with insulin (100 nM) for 15 min. The collected cells were treated as described in A . E GSK8612 attenuated the interaction between TBK1 and mTOR. HEK293T cells expressing the indicated proteins were incubated with GSK8612 (2 μM) or Torin 1 (100 nM) for 50 min prior to the cells being lysed. F MARCH1 decreased the interaction between TBK1 and mTOR. HEK293T cells expressing the indicated proteins were treated as described in B . G MARCH1 decreased the interaction between TBK1 and S6K1. HEK293T cells expressing the indicated proteins were treated as described in B . H H151 decreased the interaction of TBK with mTOR or S6K1. HEK293T cells expressing EV or TBK1-flag were treated with H151 (1 μM) for 6 h, and then subjected to immunoprecipitation with flag beads to detect the indicated proteins. I The interaction between STING and mTOR was weakened by a lack of TBK1 or overexpression of MARCH1. The shNC and shTBK1 HEK293T cells expressing the indicated proteins were treated as described in B . All the experiments were repeated three times

Article Snippet: The antibodies and reagents used in the study were as follows: p-TBK1 S172 (CST, 5483S); TBK1 (Proteintech, 28397-1-AP); p-S6K1 T389 (ABclonal, AP1059); S6K1 (ABclonal, A2190); p-AKT S473 (ABclonal, AP0637); AKT1 (CST, 2938S); mTOR (CST, 2983S); MARCH1 (ImmunoWay, YT2642); DYKDDDDK-Tag (Abmart, M20008L); MYC-Tag (CST, 2276S); V5-Tag (Abclonal, AE017); HA (CST, 3724S); HA (Abclonal, AE036); GAPDH (Abmart, M20006L); β-actin (Abclonal, AC038); β-tublin (Abmart, M20005L); p-ULK1(S757) (CST, 6888); ULK1(Abclonal, A8529); SGK1(CUSABIO, CSB-PA021189LA01HU); p-SGK1(S422) (CUSABIO, CSB-PA050044); goat anti-Mouse IgG HRP (Abmart, M21001S); goat anti-rabbit IgG HRP (Abmart, M21002S); goat anti-mouse IgG AF 488 (Abmart, M21011M); goat anti-rabbit IgG AF 594 (Abmart, M21014M); goat anti-mouse IgG AF 594 (Abmart, M21013M); goat anti-rabbit IgG AF 488 (Abmart, M21012M); EGF (Solarbio, P00033); insulin (Solarbio, I8830); Rapamycin (Selleck, S1039) and H151 (MCE, HY-112693); MG132 (Millipore, 474790); Torin 1 (MCE, HY-13003); GSK8612 (MCE, HY-111941); and ZSTK474 (MCE, HY-50847).

Techniques: Expressing, Lysis, Immunoprecipitation, Transfection, Incubation, Over Expression

Journal: Cells

Article Title: Development of Personalized Therapeutic Strategies by Targeting Actionable Vulnerabilities in Metastatic and Chemotherapy-Resistant Breast Cancer PDXs

doi: 10.3390/cells8060605

Figure Lengend Snippet: Combinatorial drug administration in PDXs is a model to uncover drug sensitivity. ( A ) Cell viability (%) in response to standard therapy with Paclitaxel (PTX) at increasing concentrations or combination with IDAS 15 µM in two TP53-Wild Type (WT; left panel) or two TP53-Mutated (Mut; right panel) PDXs is reported for n = 3 experiments (mean ± SD). ( B ) Cell viability (%) due to standard therapy with PTX at increasing concentrations or combination with EVER (10 µM concentration in TN tumors; 20 µM in MBC3 and 15 µM concentration in MBC26 cells) in two ER− (left panel) or two ER+ (right panel) PDXs ( n = 3; mean ± SD). PTX response is plotted from the same experimental setting of Figure A. ( C ) 4-Hydroxytamoxifen (4-OHT) treatment at increasing concentrations in LB PDXs: MBC3 and MBC26. Combinatorial therapy is defined as combination of standard therapy with 4-OHT and alternative therapy with IDAS (15 µM) or EVER (20 µM in MBC3 and 15 µM concentration in MBC26) in n = 3 experiments (mean ± SD). ( D ) MBC3 was used for the evaluation of pathways modulation due to single or combinatorial drug administration. Inhibition of mTOR pathway due to EVER administration was defined by western blot analysis of phospho-S6K (p-S6K) and phospho-S6 (p-S6). Activation of DNA damage response and apoptosis were evaluated by γH2Ax and PARP cleavage, respectively. Mitotic arrest was evaluated by p21 and PCNA staining. Histone H3 and Vinculin were used as normalizers.

Article Snippet: Membranes were probed with the following antibodies: p21 (Santa Cruz Biotechnology, Dallas, TX, Texas), phospho-S6K (p-S6K) (Elabscience, WuHan, China), phospho-S6 (p-S6) (Cell Signaling Technology, London, UK), γH2Ax (Biolegend, San Diego, CA, USA), PCNA (Dako, Santa Clara, CA, United States) and cleaved PARP (Cell Signaling Technology, London, UK).

Techniques: Concentration Assay, Inhibition, Western Blot, Activation Assay, Staining