Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: Chronic unpredictable mild stress promotes depression-like behaviors and upregulates USP11 in mouse prefrontal cortex. (A) Schematic overview of the experimental timeline: male C57BL/6J mice underwent 1-week adaptation, followed by 4 weeks of chronic unpredictable mild stress (CUMS) and subsequent behavioral tests. (B-E) SPT, OFT, FST, TST results in control (Ctrl) and CUMS groups (n = 8, SPT, Welch's t -test, p = 0.0204; OFT, p = 0.0101; FST, p = 0.0020; TST, p = 0.0078). (F) Western blot of p-mTOR (Ser2448) (289 kDa), total mTOR (289 kDa), p-GSK3β(Ser9) (47 kDa), total GSK3β (47 kDa), and Tubulin (55 kDa) in mPFC tissue (n = 6). (G) Quantification of p-mTOR/t-mTOR and p-GSK-3β/t-GSK-3β ratios (p-mTOR, Welch's t -test, Pp= 0.0023; p-GSK-3β, p = 0.0075). (H) Western blot of USP11 (110 kDa) and Tubulin (55 kDa) in mPFC (n = 6). (I) Quantification of USP11 protein normalized to Tubulin (p = 0.002). (J) Representative immunofluorescence images for DAPI (blue, nuclear stain), USP11 (red), and merged panels in mPFC of control and CUMS mice. Scale bar: 50 μm. (K) Mean USP11 immunofluorescence intensity quantification (n = 3, p = 0.0142). Data are shown as mean ± SEM. Statistical analysis used two-tailed unpaired Student's t-test unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For protein-binding evaluation, membranes were incubated overnight at 4 °C with GSK3β protein solution, and incubated with primary antibody against GSK3β (1:500, CST) overnight at 4 °C.

Techniques: Control, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 directly interacts with GSK3β. (A) Volcano plot of proteins detected after USP11 immunoprecipitation from mouse mPFC. Log2 fold change (x-axis) shows enrichment versus control; log2 intensity (y-axis) reflects normalized quantitation in experimental samples. USP11 served as bait; GSK3β is highlighted as an interactor (log2 intensity USP11 = 22.9, log2FC = 2.38). (B) Immunoprecipitation (IP) with anti-USP11 antibody, immunoblot (IB) detection for USP11 (110 kDa) and GSK3β (47 kDa). IP with anti-GSK3β or anti-USP11 antibody. Input: whole lysate; IgG: isotype control. (C) Validation in HEK293T transfection system: lysates of vector control or Flag-USP11 transfected cells (Flag tag, 110 kDa) subjected to IP (anti-GSK3β), IB for anti-USP11. (D) Cell lysate analysis of HEK293T single His-GSK3β, single Flag-USP11, or co-transfected groups, immunoblotted for His-GSK3β (47 kDa) and Flag-USP11 (110 kDa). (E, F) Reciprocal Co-IP verification from HEK293T co-transfection. Immunoblot analysis for His and Flag tag in His-GSK3β, Flag-USP11, and co-transfected samples. (E) Lane 1: His-GSK3β group (IP-His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP- His) (F) Lane 1: His -GSK3β group (IP- His), Lane 2: Flag-USP11 group (IP-Flag), Lane 3: Co-transfection (IP-Flag). (G) Dot blot analysis showing specific binding between USP11 and GSK3β. BSA (100/200/500 ng) served as negative control, and purified USP11 (100/200/500 ng) was spotted on the same nitrocellulose membrane. After incubation with GSK3β protein solution, binding was detected by fluorescence imaging. (H) Immunofluorescence analysis of co-localization: Exogenous expression in HEK293T cells demonstrates USP11 (red) and GSK3β (green); endogenous expression verified in primary neurons. Nuclei stained with DAPI (blue), scale bar = 25 μm. (I) Fluorescence intensity profiles along linear ROIs: Gray values of USP11 (red) and GSK3β (green) measured with ImageJ. Dual-channel curves plotted in GraphPad Prism using exported data. (J) Pearson's correlation scatter plots for USP11(red) and GSK3β(green) fluorescence, generated using ScatterJ plugin for ImageJ. Pearson's r value shown. (K) Schematic of Flag-tagged USP11 fragment constructs used for pulldown mapping. (L) HEK293T cells were co-transfected with Flag-USP11 or its deletion mutant and His- GSK3β, followed by immunoprecipitation and immunoblot analysis for Flag and His. (M) Computational molecular docking predicts multiple direct contact sites between USP11 and GSK3β.

Article Snippet: For protein-binding evaluation, membranes were incubated overnight at 4 °C with GSK3β protein solution, and incubated with primary antibody against GSK3β (1:500, CST) overnight at 4 °C.

Techniques: Immunoprecipitation, Control, Quantitation Assay, Western Blot, Biomarker Discovery, Transfection, Plasmid Preparation, FLAG-tag, Co-Immunoprecipitation Assay, Cotransfection, Dot Blot, Binding Assay, Negative Control, Purification, Membrane, Incubation, Fluorescence, Imaging, Immunofluorescence, Expressing, Staining, Generated, Construct, Mutagenesis

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 regulates GSK3β ubiquitination, phosphorylation, and synaptic protein homeostasis in neural cells (A) Western blot analysis of GSK3β ubiquitination in HEK293T cells co-transfected with Flag-vector (control), Flag-USP11 (wild-type, 110 kDa), or Flag-USP11-C318S (catalytically inactive mutant). Endogenous GSK3β and phosphorylated GSK3β at Ser9 were immunoprecipitated from cell lysates using anti-GSK3β antibody, and ubiquitination levels were detected by immunoblotting with anti-ubiquitin antibody. GSK3β: 47 kDa; ubiquitin bands detected as smear. (B) Western blot analysis of GSK3β phosphorylation in three 293T cell groups: wild-type (Ctrl), stable USP11-overexpressing line generated by lentiviral transduction (USP11-OE), and USP11-overexpressing cells subjected to siRNA knockdown (USP11-OE + siUSP11). siUSP11 was transfected to silence USP11 in the stable overexpressing cell line. Whole cell lysates were analyzed for endogenous USP11 (110 kDa), phosphorylated GSK3β at Ser9 (p-GSK3β, 47 kDa), total GSK3β (47 kDa), and GAPDH (35 kDa) as loading control. Representative results from n = 3 biological replicates per group. (C) Gray value quantification of p-GSK3β/t-GSK3β in 293T cells (n = 3, F (2, 6) = 35.38, p = 0.0005). (D) Western blot analysis of USP11 (110 kDa), phosphorylated mTOR (p-mTOR, Ser2448, 289 kDa), total mTOR (289 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), and Tubulin (55 kDa) in primary neurons upon USP11 siRNA knockdown (n = 3). (E, F) Gray value quantification of p-GSK3β/t-GSK3β, and p-mTOR/t-mTOR ratios in neurons upon USP11 siRNA knockdown (n = 3, p-GSK3β, p = 0.0213, p-mTOR, p = 0.0047). (G) Immunoblot of USP11 (110 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), SYN (77 kDa), and Tubulin (55 kDa) in primary neurons infected with adeno-associated virus (AAV) (n = 3). (H, I) Gray value quantification of p-GSK3β/t-GSK3β, and SYN/Tubulin ratios in neurons transduced with vector or AAV-USP11 viruses (n = 3, p-GSK3β, p = 0.0078, SYN, Welch's t -test, p = 0.0031). (J) Representative immunofluorescence of primary neurons transduced with vector or AAV-USP11 viruses, showing DAPI (blue, nuclei), SYN (green, synaptophysin), and USP11 (magenta); merged panels display synapse integrity. Scale bar: 50 μm. (K, L) Quantitative analysis from three independent biological replicates in primary neurons transduced with vector or AAV-USP11 viruses (K) Mean USP11 immunofluorescence intensity (p = 0.0416), (L) Mean SYN immunofluorescence intensity (p = 0.0035). Data are shown as mean ± SEM. Determined by t -test (baseline comparisons) or one-way ANOVA (multiple groups) unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: For protein-binding evaluation, membranes were incubated overnight at 4 °C with GSK3β protein solution, and incubated with primary antibody against GSK3β (1:500, CST) overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Transfection, Plasmid Preparation, Control, Mutagenesis, Immunoprecipitation, Generated, Transduction, Knockdown, Infection, Virus, Immunofluorescence

Journal: Neurobiology of Stress

Article Title: USP11 drives stress-induced synaptic structural deficits and depression-like behaviors through GSK3β/mTOR signaling

doi: 10.1016/j.ynstr.2026.100791

Figure Lengend Snippet: USP11 knockout alleviates stress-induced depressive-like behaviors and associated with mTOR Signaling (A) Western blot analysis of USP11 (110 kDa), p-mTOR (Ser2448, 289 kDa), total mTOR (289 kDa), p-GSK3β (Ser9, 47 kDa), total GSK3β (47 kDa), PSD95 (95 kDa), and Tubulin (55 kDa) in mouse mPFC from wild-type (WT) and USP11 knockout (USP11 −/− ) male mice (n = 6, Tubulin as loading control). (B–E) Quantification of baseline protein band intensity in wild-type control (WT-CON) and USP11 knockout control (KO-CON) groups: (B) USP11 (relative to Tubulin, p < 0.0001), (C) p-GSK3β (relative to total GSK3β, p = 0.0072), (D) p-mTOR (relative to total mTOR, p = 0.0028), (E) PSD95 (relative to Tubulin, p = 0.0159). n = 6/group. (F–I) Behavioral results for four groups: WT-CON, KO-CON, WT-CUMS, and KO-CUMS (OFT, distance [cm], F [3, 28] = 8.234, p = 0.0004; OFT, velocity [cm/s], F [3, 28] = 8.233, p = 0.0004; FST, F [3, 28] = 8.721, p = 0.0003; TST, F [3, 29] = 5.378, p = 0.0046). n = 8/group. (J) Western blot analysis of USP11 (110 kDa), p-mTOR (Ser2448, 289 kDa), total mTOR (289 kDa), SYN (synaptophysin, 77 kDa), and Tubulin (55 kDa) in mPFC from all four groups (n = 3). (K-M) Quantification of (K) USP11 (relative to Tubulin, F (3, 8) = 139.5, p < 0.0001), (L) p-mTOR (relative to total mTOR, F (3, 8) = 8.298, p = 0.0077), (M) SYN (relative to Tubulin, F (3, 8) = 8.811, p = 0.0065). n = 3/group. (N) Schematic overview of the experimental design, including a 7-day acclimation period, a 28-day chronic unpredictable mild stress (CUMS) procedure, the rapamycin dosing regimen (3 mg/kg, i.p., three times per week; from day 14 of CUMS until 24 h before tissue collection), and the behavioral test battery in male USP11 −/− mice. (O-R) Behavioral results for three groups in USP11 −/− mice: CON + Veh, CUMS + Veh and CUMS + Rapa. (SPT, F (2, 18) = 7.019, p = 0.0056; OFT, center time [s], F [2, 18] = 8.788, p = 0.0022; OFT, velocity [cm/s], F [2, 18] = 0.09090, p = 0.9135; TST, F [2, 18] = 7.797, p = 0.0036). n = 7/group.) (T) Quantification of p-mTOR (relative to total mTOR, F (2, 6) = 38.49, p = 0.0004) Data are shown as mean ± SEM. Determined by t -test (baseline comparisons) or one-way ANOVA (multiple groups) unless otherwise indicated. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (S) Representative immunoblots of p-mTOR (Ser2448, 289 kDa), total mTOR (289 kDa) in USP11 −/− mice under the indicated conditions. (n = 3, Tubulin as loading control).

Article Snippet: For protein-binding evaluation, membranes were incubated overnight at 4 °C with GSK3β protein solution, and incubated with primary antibody against GSK3β (1:500, CST) overnight at 4 °C.

Techniques: Knock-Out, Western Blot, Control, Battery

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Western Blot analysis of P-GSK3β (S9), GSK3β, P-C-MYC T58, C-MYC & β-ACTIN in untreated control as well as MK-1775 (0.5μM & 1μM) treated OE33, SK-GT4 & OE19 cell lines. B. Western Blot analysis of P-GSK3β (S9), GSK3β, P-C-MYC T58, C-MYC & β-ACTIN in control siRNA and WEE1 siRNA treated OE33, SK-GT4 & OE19 cell lines. C. PLA to visualize C-MYC and GSK3β interaction (red spots) in untreated Control & MK-1775 treated OE19, OE33, and SK-GT4 cell lines. Captured at 40X magnification. D. Western blot analysis of WEE1, P-CDC2 Y15, C-MYC, P-GSK3β (S9), GSK3β, and β-ACTIN in empty vector as well as WEE1-CDS vector transfected OE33, SK-GT4, and OE19 cell lines. E. Western blot analysis of WEE1, P-CDC2 Y15, CDC2, C-MYC, and β-ACTIN in empty vector, WEE1-CDS-WT, and WEE1-CDS-K328A kinase dead mutant vector transfected SK-GT4 cells.

Article Snippet: C-MYC, p-C-MYC T58, P-GSK3β S9, GSK3β, PARP, CL-PARP, Caspase 3, Cl-Caspase 3, HA tag, CD133, SOX9, SOX2, CD44, OCT4, Nanog and Ki-67 antibodies were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Inhibition, Knockdown, Phospho-proteomics, Western Blot, Control, Plasmid Preparation, Transfection, Mutagenesis

Journal: Cancer letters

Article Title: WEE1 Stabilizes MYC to Promote Therapeutic Resistance in Esophageal Adenocarcinoma

doi: 10.1016/j.canlet.2026.218418

Figure Lengend Snippet: A. Western Blot analysis of P-GSK3β (S9), GSK3β, P-C-MYC T58, C-MYC & β-ACTIN in untreated control as well as MK-1775 (0.5μM & 1μM) treated OE33, SK-GT4 & OE19 cell lines. B. Western Blot analysis of P-GSK3β (S9), GSK3β, P-C-MYC T58, C-MYC & β-ACTIN in control siRNA and WEE1 siRNA treated OE33, SK-GT4 & OE19 cell lines. C. PLA to visualize C-MYC and GSK3β interaction (red spots) in untreated Control & MK-1775 treated OE19, OE33, and SK-GT4 cell lines. Captured at 40X magnification. D. Western blot analysis of WEE1, P-CDC2 Y15, C-MYC, P-GSK3β (S9), GSK3β, and β-ACTIN in empty vector as well as WEE1-CDS vector transfected OE33, SK-GT4, and OE19 cell lines. E. Western blot analysis of WEE1, P-CDC2 Y15, CDC2, C-MYC, and β-ACTIN in empty vector, WEE1-CDS-WT, and WEE1-CDS-K328A kinase dead mutant vector transfected SK-GT4 cells.

Article Snippet: C-MYC, p-C-MYC T58, P-GSK3β S9, GSK3β, PARP, CL-PARP, Caspase 3, Cl-Caspase 3, HA tag, CD133, SOX9, SOX2, CD44, OCT4, Nanog and Ki-67 antibodies were obtained from Cell Signaling Technologies (Danvers, MA).

Techniques: Inhibition, Knockdown, Phospho-proteomics, Western Blot, Control, Plasmid Preparation, Transfection, Mutagenesis