Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 activates the Wnt/β-catenin signaling pathway in mouse hippocampal neuronal HT22 cells. ( A ) The SuperTOPFlash reporter gene was transfected into HT22 cells, and the cells were treated with solvent control (DMSO) and gradient concentrations of ISX9 (2.5-40 μM) for 24 h. Subsequently, the cells were lysed, and the fluorescence intensity was detected and normalized based on the value of the β-gal. ( B ) HT22 cells were treated with control and gradient concentrations of ISX9 (2.5-40 μM) for 24 h. The expression levels of endogenous β-catenin and activated β-catenin proteins were detected by Western blot. Quantitative analysis was performed by ImageJ and normalized relative to GAPDH values, and results were presented on the right. ( C ) HT22 cells were treated with control and 20 μM ISX9 for 24 h, respectively, followed by precipitation of cell lysates with an anti-AXIN1 antibody. The interaction of AXIN1 with LRP6 or β-catenin was detected by immunoblotting. ( D ) HT22 cells were treated with control or gradient concentrations of ISX9 (2.5-20 μM) for 24 h, respectively. After cell lysis, total RNA was extracted, and cDNA was obtained by reverse transcription. RT-qPCR was performed to detect the mRNA expression levels of Wnt target genes NOTUM, AXIN2 and SURVIVIN. The data shown are the mean ± SD (n = 3). Statistical significance was tested by one-way ANOVA followed by the Tukey test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated that there was a significant difference compared with the control group

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Transfection, Solvent, Control, Fluorescence, Expressing, Western Blot, Lysis, Reverse Transcription, Quantitative RT-PCR

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 enhances the proliferation, and promotes the expression of stemness-related Wnt target genes in hippocampal neuronal cells. HT22 cells were treated with control or gradient concentrations of ISX9 for 24 h, respectively. The cell viability ( A ) and proliferation ( B ) of HT22 cells were detected by MTT and BrdU assays. ( C ) HT22 cells were treated with control or ISX9 (2.5-20 μM) for 24 h, respectively. The cells were lysed to extract total RNA, which was then subjected to reverse transcription for cDNA synthesis. RT-qPCR was performed to detect the mRNA expression levels of stemness-related Wnt target genes LGR5 and SOX2. ( D - F ) HT22 cells were treated with control and 20 μM of ISX9 for 24 h. Then the cells were fixed and followed by IF staining with DAPI and anti-β-catenin antibody ( D ) or anti-LGR5 antibody ( E ) or anti-SOX2 antibody ( F ). Alexa Fluor 594 conjugated goat anti-mouse IgG antibodies were used as secondary antibodies. Scale bar, 20 μm. The results of the quantitative fluorescence analysis were presented on the right. The data shown are the mean ± SD (n = 3). Statistical significance was tested by one-way ANOVA followed by the Tukey test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicated that there was a significant difference compared with the control group

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Expressing, Control, Reverse Transcription, cDNA Synthesis, Quantitative RT-PCR, Staining, Fluorescence

Journal: Alzheimer's Research & Therapy

Article Title: ISX9 activates the Wnt/β-catenin signaling pathway and exerts neuroprotective effects in Alzheimer’s disease

doi: 10.1186/s13195-026-01961-5

Figure Lengend Snippet: ISX9 activates Wnt/β-catenin signaling in the hippocampus of AD mice. ( A ) Expression levels of total β-catenin and activated β-catenin protein in hippocampal tissues of WT, WT/ISX9, AD and AD/ISX9 group mice. The below bars show the results of quantification of the immunoblotting images by ImageJ and normalization relative to GAPDH values. ( B ) Protein was extracted from the hippocampal tissues of mice, followed by precipitation with an anti-AXIN1 antibody. The interaction of AXIN1 with LRP6 or β-catenin was detected by immunoblotting. ( C ) Total RNA extracted from mouse hippocampal tissues of mice was reverse transcribed to obtain cDNA, and then RT-qPCR was performed to detect the mRNA expression levels of Wnt target genes NOTUM, AXIN2 and SURVIVIN

Article Snippet: Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes for immunoblotting with the following antibodies: anti-β-catenin (Santa Cruz Biotechnology, Cat#sc-7963), anti-active β-catenin (Cell Signaling Technology, Cat#8814), anti-GAPDH (Transgen Biotech, Cat#HC301), anti-APP (Proteintech, Cat#27320-1-AP), anti-BACE1 (Proteintech, Cat#12807-1-AP), anti-Aβ (Proteintech, Cat#25524-1-AP), anti-Tau (Proteintech, Cat#10274-1-AP), anti-Phospho-Tau (S404) (Proteintech, Cat#81383-1-RR), anti-ZO-1 (Cell Signaling Technology, Cat#5406), anti-OCCLUDIN (Proteintech, Cat#5506), anti-GLUT1 (Proteintech, Cat#21829-1-AP), anti-LGR5 (Abcam, Cat#ab75732), and anti-SOX2 (Cell Signaling Technology, Cat#23064).

Techniques: Expressing, Western Blot, Reverse Transcription, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA activates Src kinase in A549 cells, but reduces the activity of recombinant Src kinase in a cell-free assay. (A) A549 cells were treated with AcoA for 1 to 24 h, lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, ** p < 0.01, *** p < 0.001 in comparison to cells incubated with vehicle for the indicated time. (B) AcoA, ouabain and digoxin reduce Src kinase activity in a cell-free assay. Enzymatic activity of recombinant c-Src kinase (0.1 U/mL) pretreated with AcoA, ouabain, digoxin, doxorubicin, or the positive control staurosporine (all 100 nM) for 30 min, was analyzed by a kinase assay. Data are mean ± SD of triplicates, * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to the vehicle control. (C) Src activation ( p -Src Y416 expression) in the NCI-60 tumor cell lines does not correlate with AcoA cytotoxicity (GI 50 values) in the same cell lines (original data were obtained from NCI database). Red dot indicates A549 cell line. Pearson correlation test. (D) AcoA and ouabain-induced cell toxicity is unaffected by the Src inhibitor PP2. Cell viability of A549 cells pretreated for 4 h with the Src kinase family inhibitor PP2 and incubated for 48 h with AcoA or ouabain (100 nM). XTT assay, mean ± SEM of N = 3.

Article Snippet: To measure Src kinase phosphorlyation, we used an ELISA Kit (PathScan Phospho-Src (Tyr416) Sandwich ELISA Kit, Cell Signaling Technology).

Techniques: Activity Assay, Recombinant, Cell-Free Assay, Activation Assay, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Comparison, Incubation, Positive Control, Kinase Assay, Control, Expressing, XTT Assay

Journal: Frontiers in Pharmacology

Article Title: The Cardenolide Glycoside Acovenoside A Interferes with Epidermal Growth Factor Receptor Trafficking in Non-Small Cell Lung Cancer Cells

doi: 10.3389/fphar.2021.611657

Figure Lengend Snippet: AcoA leads to endosomal EGFR arrest and inhibits EGF-induced degradation of EGFR (A) AcoA induces endosomal arrest in EGFR biosensor cells. After 1 h preincubation with monensin (1 µM), AcoA, digoxin, doxorubicin (all 100 nM), or vehicle, cells were stimulated with 100 ng/ml EGF and the formation of green fluorescent vesicles indicating activated and internalized EGFR was monitored microscopically. Representative images are shown. The graphs on the right show the number of fluorescent vesicles/cell at the respective time point. Data are mean ± SEM of N = 3 independent experiments, *** p < 0.001. (B) Quantification of cellular EGFR in A549 cells treated as in (A) using a commercial ELISA In-cell ELISA assay. The amount of EGFR was normalized to cell number assessed by crystal violet staining. Data are mean ± SEM of N = 3 independent experiments, * p < 0.05 vs. control, # p < 0.05 vs. EGF treatment group. (C) At the same conditions, EGFR activation was assessed using ELISA for the Y1173 phosphorylation site of EGFR. EGFR phosphorylation is expressed as the ratio of phosphorylated EGFR to total EGFR. Data are mean ± SEM of N = 3, * p < 0.05 vs. control. (D) Src kinase activation was measured as a downstream target of EGFR. A549 cells treated as in (A) were lysed and the cell lysates were analyzed for activation of the Tyr416 phosphorylation site of Src kinase using a commercial ELISA assay. Data are mean ± SEM of N = 3, * p < 0.05 vs. control, ** p < 0.01 vs. control, # p < 0.05 vs. EGF treatment group.

Article Snippet: To measure Src kinase phosphorlyation, we used an ELISA Kit (PathScan Phospho-Src (Tyr416) Sandwich ELISA Kit, Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, In-Cell ELISA, Staining, Control, Activation Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Heterogeneous Sensitivity to Src Inhibitors in Oral Squamous Cell Carcinoma and Its Implications for Combination Therapy with Cisplatin

doi: 10.64898/2026.04.02.716058

Figure Lengend Snippet: A Western blot analysis of the oral squamous carcinoma cell lines to monitor numerous signaling pathways. The phosphorylated and total protein levels were determined as indicated. B Cell viability (%) of the oral squamous carcinoma cell lines treated with each Src inhibitor for 72 h. Viability was measured using CellTiter-Glo, and values are indicated relative to the 1% dimethyl sulfoxide (DMSO) control. Data represent mean ± standard deviation from three independent experiments (n = 3). C IC50 curves of the oral squamous carcinoma cell lines treated with each Src inhibitor for 36 h. Viability was measured using CellTiter-Glo and normalized to that of the 1% DMSO control. Values represent the mean of n = 3. D, E Western blot analysis of the oral squamous carcinoma cell lines to investigate Src (D) and MAPK (E) activity induced by different Src inhibitors. Oral squamous carcinoma cell lines were treated with an Src inhibitor for 18 h, followed by western blot analysis. D indicates the Src phosphorylation (Tyr416), and E reveals MAPK pathway-related proteins. Supplementary Table S1 lists the concentrations of the Src inhibitors used.

Article Snippet: The following primary antibodies were used: Phospho-Src Family (Tyr416) (D49G4) Rabbit mAb (#6943), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (#4370), Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP Rabbit mAb (#4511), and Phospho-SAPK/JNK (Thr183/Tyr185) (81E11) Rabbit mAb (#4668) (all from Cell Signaling Technology).

Techniques: Western Blot, Protein-Protein interactions, Control, Standard Deviation, Activity Assay, Phospho-proteomics