ma 104  (ATCC)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Integrated phenotypic screening and chemical proteomics identifies ETF1 ligands that modulate viral translation and replication

doi: 10.1073/pnas.2524108123

Figure Lengend Snippet: Photo-stereoprobes inhibit additional viruses that use PRF. ( A ) Effect of active stereoprobe WX-02-31 and control stereoprobes on HIV-1 pseudovirus production. ( Top ) Schematic of the HIV-1 ribosomal frameshift sequence. ( Bottom ) HEK293T cells expressing the HIV-1 NL4-3 luciferase vector were treated with the indicated photo-stereoprobes (10 µM) and assessed for luciferase activity after 24 h. Data are average values ± SD for 2 to 5 independent experiments that each have three technical replicates. ( B ) Effect of active stereoprobe WX-02-31 and control stereoprobes on Sindbis virus (SINV) infection. ( Top ) Schematic of the SINV ribosomal frameshift sequence. ( Bottom ) HeLa cells were pretreated with indicated photo-stereoprobes (10 µM, 3 h), inoculated with SINV (MOI 1) in the presence of compounds, and viral supernatant titers were assessed after 48 hpi. Data are average values ± SD for three independent experiments that each have 2 to 3 technical replicates. ( C ) Effect of active stereoprobe WX-02-31 and control stereoprobes on porcine reproductive and respiratory syndrome virus (PRRSV) infection. ( Top ) Schematic of the PRRSV ribosomal frameshift sequence. ( Bottom ) MA-104 cells were pretreated with indicated photo-stereoprobes (10 µM, 3 h), inoculated with PRRSV-GFP (MOI 0.1) in the presence of compounds, and viral antigen levels were assessed by GFP expression after 24 hpi. Data are representative of two independent experiments that each have three technical replicates. ( D ) Effect of active stereoprobe WX-02-31 and control stereoprobes on lymphocytic choriomeningitis virus (LCMV) infection. ( Top ) Schematic of the LCMV genome. ( Bottom ) HeLa cells were pretreated with indicated photo-stereoprobes (10 µM, 3 h), inoculated with LCMV (clone 13, MOI 0.01) in the presence of compounds and viral supernatant titers were assessed after 48 hpi. Data are average values ± SD for two independent experiments that each have 2 to 3 technical replicates. ( E ) Effect of active stereoprobe WX-02-31 and control stereoprobes on influenza A virus (IAV) infection. ( Top ) Schematic of the IAV genome. ( Bottom ) Calu-3 cells were pretreated with indicated photo-stereoprobes (20 µM, 3 h), inoculated with IAV (MOI 0.005) in the presence of compounds, and viral antigen levels were assessed by flow cytometry after 48 hpi. Data are the average values ± SD for two independent experiments that each have three technical replicates. For ( A – C ), two-tailed unpaired t test comparing WX-02-31 versus DMSO or WX-02-51 groups: * P < 0.05, **** P < 0.0001.

Article Snippet: Calu-3 (HTB-55), Vero E6 (CRL-1586), HEK-293T (CRL-3216), HeLa (CCL-2), NIH-3T3 (CRL-1658), MA-104 (CRL-2378.1), and MDCK (CCL-34) cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Sequencing, Expressing, Luciferase, Plasmid Preparation, Activity Assay, Virus, Infection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: A GluN2B disease-associated variant promotes the degradation of NMDA receptors via autophagy

doi: 10.1016/j.jbc.2026.111147

Figure Lengend Snippet: The GluN2B subunit undergoes degradation via autophagy . A , inhibition of the proteasome with MG132 (10 μM) and the lysosome with Bafilomycin-A1 (1 μM) for 6 h effect on the GluN2B subunit in HEK293T cells stably expressing recombinant WT or R519Q NMDARs. β-actin serves as the soluble total protein loading control (n = 5). B , effects of R519Q on total GluN2B protein expression levels 48 h post transient transfection with GluN1 and GluN2B constructs at a 1:1 ratio in ATG7 KO HEK293T to express WT or GluN2B_R519Q variant NMDARs. β-actin served as the soluble total protein loading control. (n = 6). C , surface biotinylation assay to monitor the influence of the R519Q DAV on the surface expression of NMDARs expressed in ATG7 KO HEK293T 48 h post transient transfection. Na + /K + ATPase served as a membrane protein loading control (n = 6). D , schematic of macroautophagy pathway, including induction, nucleation and formation of the isolation membrane, elongation, autophagosome formation, and fusion with the lysosome forming the autophagolysosome. Rapamycin is shown to inhibit mTOR, which results in autophagy activation, while 3-MA and Baf-A1 inhibit autophagy at different stages as shown. E , HEK293T cells stably expressing R519Q NMDARs were treated with autophagy activators (SMER28 10 μM, Rapamycin 100 nM) and inhibitors of autophagy (3-MA 50 mM and Baf-A1 20 nM) for 24 h. Changes to total GluN2B expression were monitored via immunoblot and p62 was used as a marker for autophagic flux. β-actin serves as the soluble total protein loading control (n = 3). F , siRNA knockdown of ATG7 effect on R519Q variant GluN2B expression and immunoblot was performed 48 h after knockdown. β-actin served as the soluble total protein loading control (n = 3). Non-targeting (NT) siRNA was used as a control for each condition, and two independent siRNA constructs were used to knockdown gene expression. G , siRNA knockdown of LC3b effects on R519Q variant GluN2B expression. Immunoblot performed 48 h after knockdown. β-actin served as the soluble total protein loading control (n = 3). Non-targeting siRNA was used as a control for each condition, and two independent siRNA constructs were used to knockdown gene expression. LC3-Pool contains two unique siRNAs for each of the LC3a, LC3b, and LC3c isoforms. All data are normalized to the appropriate loading control, and data are presented as mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test between two groups or an analysis of variance (ANOVA) followed by a post hoc Dunnett’s test for comparison in multiple groups. Significance level defined as ns, not significant, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: The 3-methyladenine (3-MA) (#HY-19312), SMER28 (#HY-100200), rapamycin (#HY-10219), and DAPI dihydrochloride (HY-D0814) were obtained from MedChemExpress.

Techniques: Inhibition, Stable Transfection, Expressing, Recombinant, Control, Transfection, Construct, Variant Assay, Surface Biotinylation Assay, Membrane, Isolation, Activation Assay, Western Blot, Marker, Knockdown, Gene Expression, Two Tailed Test, Comparison

Journal: Translational Oncology

Article Title: Zeaxanthin targets TOP2A to regulate autophagy and suppress lung cancer progression via the MAPK/ERK pathway

doi: 10.1016/j.tranon.2025.102658

Figure Lengend Snippet: TOP2A promotes LC progression by inhibiting cellular autophagy. A-B: Molecular docking diagrams 2D (A) and 3D (B) of Zea and TOP2A; C: The effect of Zea on the thermal stability of TOP2A analyzed by CETSA; A549 cells: NC group, ETOH group, Zea group; D: TOP2A protein levels detected by WB; A549 cells: si-NC, si-TOP2A, E: TOP2A mRNA levels detected by qRT-PCR; F: TOP2A protein levels detected by WB; A549 cells: si-NC, si-TOP2A, si-TOP2A+3-MA, G: Cell viability detected by CCK-8; H: Cell proliferation detected by colony formation assay; I: Cell migration detected by Transwell; J: Cell apoptosis detected by flow cytometry. ** P < 0.01, *** P < 0.001. The data is presented as mean ± standard deviation, and inter-group comparisons are conducted using ANOVA and Tukey’s post hoc test.

Article Snippet: After successful transfection of TOP2A was confirmed by qRT-PCR and WB ( E– ), the autophagy inhibitor 3-MA (MCE, USA) was added to set the rescue experiment.

Techniques: Quantitative RT-PCR, CCK-8 Assay, Colony Assay, Migration, Flow Cytometry, Standard Deviation