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