3 methyladenine 3 ma  (MedChemExpress)

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

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: Effects of PT on chondrocyte viability and ECM metabolism under IL-1β stimulation. (A,B) Cell viability determined by CCK-8 assay in chondrocytes treated with increasing PT concentrations (0, 5, 10, 20, 40, 100 μM) for 24 and 48 h without (A) or with (B) IL-1β (10 ng/mL) stimulation; (C) Representative WB images showing ACAN, COL2A1, MMP3 and MMP13 expression; (D–G) Quantitative analysis of protein levels normalized to GAPDH for ACAN (D) , COL2A1 (E) , MMP13 (F) , and MMP3 (G) . Data = mean ± SEM (n = 3). *P < 0.05, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. IL-1β.

Article Snippet: In cell viability assays, cells were exposed to PT at concentrations of 0, 5, 10, 20, 40, and 100 μM, with or without interleukin-1β (IL-1β, 10 ng/mL, HY-P7028, MedChemExpress, Shanghai, China), for 24 and 48 h. To assess PT’s effects on ECM metabolism, cells were treated with IL-1β (10 ng/mL) alone or in combination with PT (10, 20, or 40 μM) for 24 h. To investigate the role of autophagy in PT-mediated chondroprotection, cells were divided into five groups: control (vehicle), IL-1β (10 ng/mL), IL-1β + PT (20 μM), IL-1β + rapamycin (RAPA, 100 nM; HY-10219, MedChemExpress, Shanghai, China), and IL-1β + PT + 3 MA (3-methyladenine, 5 mM; HY-19312, MedChemExpress, Shanghai, China).

Techniques: CCK-8 Assay, Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: PT protects chondrocytes against IL-1β-induced damage via autophagy activation. (A) Representative WB images showing ACAN, COL2A1, MMP3, MMP13, Beclin1, LC3B, and p62 expression; (B–H) Quantitative analysis of protein levels normalized to GAPDH for ACAN (B) , COL2A1 (C) , MMP13 (D) , MMP3 (E) , Beclin1 (F) , LC3II/I ratio (G) , and p62 (H) ; (I) Representative TUNEL staining showing apoptotic cells (red) and nuclei (blue) (scale bar = 50 μm); (J) Percentage of TUNEL-positive cells. Data = mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 vs. control; ###P < 0.001 vs. IL-1β; †P < 0.05, †††P < 0.001 vs. IL-1β + PT.

Article Snippet: In cell viability assays, cells were exposed to PT at concentrations of 0, 5, 10, 20, 40, and 100 μM, with or without interleukin-1β (IL-1β, 10 ng/mL, HY-P7028, MedChemExpress, Shanghai, China), for 24 and 48 h. To assess PT’s effects on ECM metabolism, cells were treated with IL-1β (10 ng/mL) alone or in combination with PT (10, 20, or 40 μM) for 24 h. To investigate the role of autophagy in PT-mediated chondroprotection, cells were divided into five groups: control (vehicle), IL-1β (10 ng/mL), IL-1β + PT (20 μM), IL-1β + rapamycin (RAPA, 100 nM; HY-10219, MedChemExpress, Shanghai, China), and IL-1β + PT + 3 MA (3-methyladenine, 5 mM; HY-19312, MedChemExpress, Shanghai, China).

Techniques: Activation Assay, Expressing, TUNEL Assay, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene attenuates osteoarthritis progression through p53-dependent autophagy activation: evidence from network analysis and experimental validation

doi: 10.3389/fphar.2026.1686555

Figure Lengend Snippet: PT modulates p53 localization, AMPK/mTOR signaling, and autophagy-related markers in chondrocytes. (A) CETSA results assessing the thermal stability of p53 in C28/I2 cells treated with PT or vehicle across a temperature range of 38 °C–70 °C. Representative WB bands are shown above the normalized thermal aggregation curves; (B) Representative immunofluorescence confocal microscopy images illustrating p53 subcellular localization (red) and DAPI-stained nuclei (blue) in chondrocytes across different treatment groups (scale bar = 10 μm); (C) Representative WB bands showing p53 distribution (nuclear, cytoplasmic, and total), the phosphorylation status of AMPK and mTOR, and autophagy markers (LC3 and p62); (D–K) Quantitative analyses of protein expression levels: total p53/GAPDH (D) , nuclear p53/PCNA (E) , cytoplasmic p53/GAPDH (F) , nuclear/cytoplasmic p53 ratio (G) , p-AMPK/AMPK (H) , p-mTOR/mTOR (I) , p62/GAPDH (J) , and the LC3II/I ratio (K) ; (L) Representative TUNEL staining images showing apoptotic cells (red) and nuclei (blue) (scale bar = 50 μm); (M) Quantitative analysis of the percentage of TUNEL-positive cells across groups. Data = mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 vs. control; ###P < 0.001 vs. IL-1β; †P < 0.05, ††P < 0.01, †††P < 0.001 vs. IL-1β + PT.

Article Snippet: In cell viability assays, cells were exposed to PT at concentrations of 0, 5, 10, 20, 40, and 100 μM, with or without interleukin-1β (IL-1β, 10 ng/mL, HY-P7028, MedChemExpress, Shanghai, China), for 24 and 48 h. To assess PT’s effects on ECM metabolism, cells were treated with IL-1β (10 ng/mL) alone or in combination with PT (10, 20, or 40 μM) for 24 h. To investigate the role of autophagy in PT-mediated chondroprotection, cells were divided into five groups: control (vehicle), IL-1β (10 ng/mL), IL-1β + PT (20 μM), IL-1β + rapamycin (RAPA, 100 nM; HY-10219, MedChemExpress, Shanghai, China), and IL-1β + PT + 3 MA (3-methyladenine, 5 mM; HY-19312, MedChemExpress, Shanghai, China).

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Phospho-proteomics, Expressing, TUNEL Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Host E3 ligase RNF2 restricts African swine fever virus replication through targeting viral pA104R for its K5 site ubiquitination degradation

doi: 10.1007/s00018-026-06085-5

Figure Lengend Snippet: RNF2 mediates the degradation of pA104R via both the proteasome and autophagy pathways. ( A ) HEK-293T cells were cotransfected with pA104R-HA (1 µg) and increasing doses (0, 0.25, 0.5, 1, 2, and 4 µg) of the RNF2-Flag plasmid for 48 h. ( B ) as a control, p72-HA and increasing doses of the RNF2-Flag plasmid were also cotransfected into HEK-293T cells for 48 h. The cells were collected at 48 hpt, and the protein expression levels of pA104R, p72 and RNF2 were measured via Western blotting. ( C ) 3D4/21 cells were transfected with increasing doses (0.25, 0.5, 0.75, or 1 µg) of the RNF2-Flag plasmid for 24 h before ASFV CN/SC/2019 infection (MOI = 1) for 24 h. The cells were subsequently collected, and the protein expression levels of pA104R and RNF2 were measured via Western blotting. ( D ) pA104R-HA was cotransfected with or without RNF2-Flag into HEK-293T cells, which were then treated with DMSO (negative control), MG-132 (10 µM), NH4Cl (20 mM), or 3-MA (5 mM) for 24 h. The cells were then collected, and the protein expression levels of pA104R and RNF2 were measured via Western blotting

Article Snippet: MG-132 (HY-13259), Z-VAD-FMK (HY-16658B) and 3-MA (HY-19312) were purchased from MedChemExpress (MCE); DMSO (67–68-5) was purchased from Sigma‒Aldrich; Commercial ELISA kit of IL-18 (ES12RB), SYBR Green (4385612) and NP-40 (FNN0021) were purchased from Thermo Fisher Scientific.

Techniques: Plasmid Preparation, Control, Expressing, Western Blot, Transfection, Infection, Negative Control