Journal: Neurochemical Research

Article Title: Morin Improves Cognitive Deficits in an in Vivo Model of Vascular Dementia by Modulating the N-methyl-D-aspartate Receptor Signaling Pathways

doi: 10.1007/s11064-026-04717-7

Figure Lengend Snippet: Morin modulated the expression of NMDA receptors in the hippocampus of VaD rats. a the expression levels of NR1 ; b the expression levels of NR2A ; c the expression levels of NR2B ; d the expression levels of NR1 protein; e the expression levels of NR2A protein; f the expression levels of NR2B protein; g protein levels of p-CREB; h protein levels of p-CAMK2A; i protein levels of p-CAMK2D. Protein levels of NR1, NR2A, and NR2B were quantified by ELISA. Data are presented as mean ± SD ( n = 8 per group). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc test (data met assumptions of normality and homoscedasticity)/Kruskal-Wallis with Dunn’s test (data did not meet assumptions). * indicates a significant difference from the Sham group; # indicates a significant difference 2VO group; *# indicates a significant difference from both the Sham and 2VO groups, with a p-value of less than 0.05 considered statistically significant

Article Snippet: Moreover, phosphorylation levels of calcium/calmodulin-dependent protein kinase II isoforms CAMK2A and CAMK2D at Thr286 (p-CAMK2A, p-CAMK2D) were quantified using the Colorimetric Cell-Based ELISA Kit (CAMK2A/CAMK2D (Phospho-Thr286), Boster Bio, #EKC2366).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Clinical Interventions in Aging

Article Title: Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington’s disease via suppressing oxidative stress, inflammation, and apoptosis

doi: 10.2147/cia.s86493

Figure Lengend Snippet: Figure 7 Expressions of calpain and caspase 12, two markers of ER stress-related apoptosis, in the heart tissue of R6/2 HD mice under oral B307 treatment, and of their WT. Notes: (A) IHC staining illustrates that expressions of calpain and caspase 12 (shown in brown) were remarkable in comparison to their WT but were reduced under oral B307 treatment. Scale bars: 25 µm. (B) Western blotting analysis shows the following: (a) Cardiac expression levels of calpain and caspase 12 in R6/2 HD mice under sham and oral B307 treatments, and of their WT. (b) Quantified cardiac calpain and caspase 12 levels in R6/2 HD mice were significantly enhanced in comparison to their WT but were significantly weaker under oral B307 treatment. There were six mice per each group. Values are mean ± SEM (**P,0.01, *P,0.05, two-way ANOVA followed by a Student–Newman–Keuls multiple comparisons posttest). Abbreviations: WT, wild-type littermate controls; HD, Huntington’s disease; ER, endoplasmic reticulum; IHC, immunohistochemical; SEM, standard error of the mean; ANOVA, analysis of variance.

Article Snippet: This study used antibodies such as b-actin (Thermo Fisher Scientific Inc.), TNF-α (Cat #11948, Cell Signaling Technology Inc.), SOD2 (Cat #13141, Cell Signaling Technology Inc.), 3-NT (Cat ab61392, Abcam Inc.), 4-HNE (Cat ab46545, Abcam Inc.), Bcl-2 (Cat sc-7382, Santa Cruz Biotechnology Inc.), Bax (Cat #MS-714, Thermo Fisher Scientific Inc.), Cyto-C (Cat #1896-1, Abcam Inc.), calpain (Cat #2539, Cell Signaling Technology Inc.), caspase 12 (Cat #2202, Cell Signaling Technology Inc.), caspase 9 (Cat #9504, Cell Signaling Technology Inc.), and caspase 3 (Cat #9662, Cell Signaling Technology Inc.) to identify expression levels of these proteins in cardiac tissue by means of a horseradish peroxidase-linked secondary antibody.

Techniques: Immunohistochemistry, Comparison, Western Blot, Expressing, Immunohistochemical staining

Journal: Clinical Interventions in Aging

Article Title: Oral treatment with herbal formula B307 alleviates cardiac failure in aging R6/2 mice with Huntington’s disease via suppressing oxidative stress, inflammation, and apoptosis

doi: 10.2147/cia.s86493

Figure Lengend Snippet: Figure 9 The schematic diagram illustrates the possible cardioprotective pathways under oral B307 treatment. Notes: Cardioprotection in R6/2 HD mice under B307 treatment may be promoted via attenuating oxidative stress (marked by increasing expression of SOD2), anti- apoptosis (marked by increasing expression of Bcl-2), reducing mHtt aggregation, oxidative stress (marked by reducing expressions of 3-NT and 4-HNE), inflammation (marked by reducing expression of TNF-α), ER stress-related apoptosis (marked by reducing expressions of calpain and caspase 12), and mitochondrial dysfunction-related apoptosis (marked by reducing expressions of Bax, Cyto-C, caspase 9, and caspase 3) in heart tissue. Abbreviations: mHtt, mutant huntingtin; SOD2, superoxide dismutase 2; ROS, reactive oxygen species; 3-NT, neurotrophin-3; 4-HNE, 4-hydroxynonenal; TNF-α, tumor necrosis factor alpha; ER, endoplasmic reticulum; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; Cyto-C, cytochrome C; ER, endoplasmic reticulum; WT, wild-type littermate controls; HD, Huntington’s disease.

Article Snippet: This study used antibodies such as b-actin (Thermo Fisher Scientific Inc.), TNF-α (Cat #11948, Cell Signaling Technology Inc.), SOD2 (Cat #13141, Cell Signaling Technology Inc.), 3-NT (Cat ab61392, Abcam Inc.), 4-HNE (Cat ab46545, Abcam Inc.), Bcl-2 (Cat sc-7382, Santa Cruz Biotechnology Inc.), Bax (Cat #MS-714, Thermo Fisher Scientific Inc.), Cyto-C (Cat #1896-1, Abcam Inc.), calpain (Cat #2539, Cell Signaling Technology Inc.), caspase 12 (Cat #2202, Cell Signaling Technology Inc.), caspase 9 (Cat #9504, Cell Signaling Technology Inc.), and caspase 3 (Cat #9662, Cell Signaling Technology Inc.) to identify expression levels of these proteins in cardiac tissue by means of a horseradish peroxidase-linked secondary antibody.

Techniques: Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

doi: 10.1101/2024.02.29.582877

Figure Lengend Snippet: ( A ) Treatment with IL6 (20 ng/mL) for 2 hours (left panel) or the induction of cancer stem cells (CSCs) (right panel) upregulates the expression of most glycolysis-related genes, including PFKL, at the mRNA level. Red bars represent PLC5 cells after IL6 treatment or sphere formation, while black bars represent control cells. ( B ) IL6 treatment increases PFKL protein levels. The right panel shows the quantification results of western blot analysis for PFKL and RPIA protein levels. Black bars indicate no treatment control, red bars represent 0.5 hours of IL6 treatment, and blue bars represent 2 hours of IL6 treatment. ( C ) Knockdown of AMPK by shRNA reduces IL6-induced PFKL-upregulated protein expression in PLC5 cells, leading to decreases in pAMPK, PFKL, RPIA, pERK, and pSMAD5 levels, while it has no effect on STAT3 phosphorylation. The right panel illustrates the quantification of western blot results for AMPK, pAMPK, PFKL, RPIA, STAT3, pSTAT3, ERK, pERK, mTOR, p-mTOR, SMAD5, and pSMAD5. Black bars represent no treatment control, red bars indicate IL6 treatment, blue bars represent shAMPK, and the green bar denotes shAMPK+IL6. ( D ) Knockdown of AMPK decreases cell viability in PLC5 cells. Quantification of cell viability at 24, 48, and 72 hours is shown. Red bars represent shAMPK, while black bars denote shLuc control. ( E ) AMPK knockdown reduces IL6-stimulated cell viability, normalized to the control without IL6 treatment. Black bars represent the control without treatment, and red bars represent IL6 treatment. ( F ) Inhibition of AMPK with dorsomorphin decreases cell viability in three hepatoma cell lines. Cell death rates were quantified, and the IC50 for dorsomorphin in PLC5, Hep3B, and HepG2 cells is displayed in the upper left. ( G ) AMPK knockdown decreases migration ability with or without IL6 treatment. Quantification of migration without IL6 or with IL6 treatment is shown. Black bars represent sh-Luc control, and red bars denote sh-AMPK. ( H ) Suppression of AMPK with dorsomorphin reduces migration ability. Black bars represent the control without treatment, and red bars indicate dorsomorphin treatment. ( I ) Dorsomorphin significantly reduces PFKL protein levels. The left panel presents quantification of western blot results for AMPK, pAMPK, PFKL, SMAD5, pSMAD5, ACC, and pACC. Black bars represent the control without treatment, light red bars denote 1 µg/mL, and red bars indicate 6 µg/mL dorsomorphin treatment. ( J ) AMPK knockdown does not affect PFKL mRNA expression. Quantification of qPCR results for PFKL mRNA is shown. The red bar indicates dorsomorphin treatment, while the black bar denotes the control without treatment. ( K ) AMPK knockdown increases proteasome activity in PLC5 cells, suggesting that AMPK stabilizes PFKL by inhibiting proteasome activity. Quantification of proteasome activity is shown, with the red bar indicating shAMPK and the black bar representing shLuc control. Statistical analyses were performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The specific antibodies against PFKL (Cell Signaling Technology Cat# 8175, RRID:AB_11178807), STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588), pSTAT3 (Cell Signaling Technology Cat# 9130, RRID:AB_330367), AMPK (Cell Signaling Technology Cat# 2603, RRID:AB_490795), pAMPK (Cell Signaling Technology Cat# 5759, RRID:AB_10949320), ERK (GeneTex Cat# GTX59618, RRID:AB_10726211), pERK (Abcam Cat# ab32538, RRID:AB_11156273), RPIA (Abcam Cat# ab67080, RRID:AB_1142656), PK (GeneTex Cat# GTX111536, RRID:AB_1951258), G6P (GeneTex Cat# GTX113203, RRID:AB_2037119), TPI (GeneTex Cat# GTX104618, RRID:AB_1241405), pRaf (BioVision Cat# 3504-100, RRID:AB_2060496), pMEK1/2 (Cell Signaling Technology Cat# 9121, RRID:AB_331648), pSMAD5 (Abcam Cat# ab92698, RRID:AB_10561456), SMAD5 (Abcam Cat# ab40771, RRID:AB_777981), pACC (Cell Signaling Technology Cat# 3661, AB_330337), ACC (Cell Signaling Technology Cat# 3662, RRID:AB_2219400), pmTOR(Cell Signaling Technology Cat#2974, RRID: AB_2262884), mTOR (Cell Signaling Technology Cat#2983, RRID: AB_ 2105622), α/β-Tubulin (Cell Signaling Technology Cat#2148, RRID: AB_2288042), GAPDH (GeneTex Cat# GTX100118, RRID:AB_1080976), β-actin (GeneTex Cat# GTX109639, RRID:AB_1949572) and ubiquitin (Cell Signaling Technology Cat# 3936, RRID:AB_331292) were purchased from Cell Signaling (Danvers, Massachusetts, USA), Abcam (Cambridge, Massachusetts, USA), and GeneTex (Irvine, CA, USA).

Techniques: Expressing, Control, Western Blot, Knockdown, shRNA, Phospho-proteomics, Inhibition, Migration, Activity Assay

Journal: bioRxiv

Article Title: Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

doi: 10.1101/2024.02.29.582877

Figure Lengend Snippet: ( A ) Treatment with the STAT3 inhibitors nifuroxazide and BBI608 reduces AMPK mRNA levels in PLC5 cells treated with IL6. ( B ) Nifuroxazide and BBI608 treatments block IL6-induced PFKL-upregulated mRNA in PLC5 cells. ( C ) Chromatin immunoprecipitation assays reveal that pSTAT3 directly binds to the promoter regions of AMPK and PFKL in IL6-treated PLC5 cells. ( D ) Inhibition of STAT3 by nifuroxazide and BBI608 diminishes the IL6-mediated increase in PFKL protein levels, as shown by Western blot analysis. The lower panel quantifies the western blot results for PFKL, STAT3, and pSTAT3 protein levels. Red bars represent nifuroxazide treatment, blue bars denote BBI608 treatment, and black bars represent the no treatment control. ( E ) Treatment with the AMPK inhibitor BBI608 reduces the levels of glycolytic and lipogenic metabolites. Red bars represent the BBI608 treatment, while black bars represent the no treatment control. ( F ) Knockdown of PFKL with shPFKL leads to a significant reduction in glycolytic and lipogenic metabolites. Red bars indicate shPFKL, and black bars represent shLuc control. ( G ) Nifuroxazide and BBI608 treatments markedly decreased glucose uptake in PLC5 cells, as indicated by a reduction in ( H ) a tumor xenograft model, detected using FDG nuclear imaging. Arrows indicate the xenograft sites. Quantification of glucose uptake at the xenograft sites is shown. Red bars represent the BBI608 treatment, while black bars denote the no treatment control. ( I ) Targeting STAT3 with nifuroxazide and BBI608 reduces the migration ability of PLC5 cells. Red bars indicate nifuroxazide treatment, blue bars denote BBI608 treatment, and black bars represent the no treatment control. ( J ) PLC5 cells cultured in spheres as cancer stem cells for 10 days under specific conditions. ( K ) PLC5 tumorspheres exhibit increased migration ability compared to parental cells. ( L ) PLC5 tumorspheres display elevated mRNA expression levels of PFKL and AMPK. ( M ) The size of PLC5 tumorspheres can be reduced by STAT3 inhibitors but not by sorafenib. ( N , O ) Knockdown of PFKL and AMPK and suppression of AMPK and STAT3 resulted in a remarkable reduction in mitochondrial respiration and ATP production. Statistical analyses were performed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: The specific antibodies against PFKL (Cell Signaling Technology Cat# 8175, RRID:AB_11178807), STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588), pSTAT3 (Cell Signaling Technology Cat# 9130, RRID:AB_330367), AMPK (Cell Signaling Technology Cat# 2603, RRID:AB_490795), pAMPK (Cell Signaling Technology Cat# 5759, RRID:AB_10949320), ERK (GeneTex Cat# GTX59618, RRID:AB_10726211), pERK (Abcam Cat# ab32538, RRID:AB_11156273), RPIA (Abcam Cat# ab67080, RRID:AB_1142656), PK (GeneTex Cat# GTX111536, RRID:AB_1951258), G6P (GeneTex Cat# GTX113203, RRID:AB_2037119), TPI (GeneTex Cat# GTX104618, RRID:AB_1241405), pRaf (BioVision Cat# 3504-100, RRID:AB_2060496), pMEK1/2 (Cell Signaling Technology Cat# 9121, RRID:AB_331648), pSMAD5 (Abcam Cat# ab92698, RRID:AB_10561456), SMAD5 (Abcam Cat# ab40771, RRID:AB_777981), pACC (Cell Signaling Technology Cat# 3661, AB_330337), ACC (Cell Signaling Technology Cat# 3662, RRID:AB_2219400), pmTOR(Cell Signaling Technology Cat#2974, RRID: AB_2262884), mTOR (Cell Signaling Technology Cat#2983, RRID: AB_ 2105622), α/β-Tubulin (Cell Signaling Technology Cat#2148, RRID: AB_2288042), GAPDH (GeneTex Cat# GTX100118, RRID:AB_1080976), β-actin (GeneTex Cat# GTX109639, RRID:AB_1949572) and ubiquitin (Cell Signaling Technology Cat# 3936, RRID:AB_331292) were purchased from Cell Signaling (Danvers, Massachusetts, USA), Abcam (Cambridge, Massachusetts, USA), and GeneTex (Irvine, CA, USA).

Techniques: Blocking Assay, Chromatin Immunoprecipitation, Inhibition, Western Blot, Control, Knockdown, Imaging, Migration, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Stabilization of AMPK/PFKL/RPIA in the Glycolytic Bodies Transduces IL6/STAT3 Signal in Hepatocarcinogenesis

doi: 10.1101/2024.02.29.582877

Figure Lengend Snippet: pSTAT3 increases the transcription of AMPK and PFKL. AMPK stabilizes the PFKL protein level. PFKL specifically stabilizes RPIA. AMPK, PFKL and RPIA are colocalized in the G-body. Inhibiting STAT3 or AMPK can reduce tumor proliferation and metastasis.

Article Snippet: The specific antibodies against PFKL (Cell Signaling Technology Cat# 8175, RRID:AB_11178807), STAT3 (Cell Signaling Technology Cat# 9132, RRID:AB_331588), pSTAT3 (Cell Signaling Technology Cat# 9130, RRID:AB_330367), AMPK (Cell Signaling Technology Cat# 2603, RRID:AB_490795), pAMPK (Cell Signaling Technology Cat# 5759, RRID:AB_10949320), ERK (GeneTex Cat# GTX59618, RRID:AB_10726211), pERK (Abcam Cat# ab32538, RRID:AB_11156273), RPIA (Abcam Cat# ab67080, RRID:AB_1142656), PK (GeneTex Cat# GTX111536, RRID:AB_1951258), G6P (GeneTex Cat# GTX113203, RRID:AB_2037119), TPI (GeneTex Cat# GTX104618, RRID:AB_1241405), pRaf (BioVision Cat# 3504-100, RRID:AB_2060496), pMEK1/2 (Cell Signaling Technology Cat# 9121, RRID:AB_331648), pSMAD5 (Abcam Cat# ab92698, RRID:AB_10561456), SMAD5 (Abcam Cat# ab40771, RRID:AB_777981), pACC (Cell Signaling Technology Cat# 3661, AB_330337), ACC (Cell Signaling Technology Cat# 3662, RRID:AB_2219400), pmTOR(Cell Signaling Technology Cat#2974, RRID: AB_2262884), mTOR (Cell Signaling Technology Cat#2983, RRID: AB_ 2105622), α/β-Tubulin (Cell Signaling Technology Cat#2148, RRID: AB_2288042), GAPDH (GeneTex Cat# GTX100118, RRID:AB_1080976), β-actin (GeneTex Cat# GTX109639, RRID:AB_1949572) and ubiquitin (Cell Signaling Technology Cat# 3936, RRID:AB_331292) were purchased from Cell Signaling (Danvers, Massachusetts, USA), Abcam (Cambridge, Massachusetts, USA), and GeneTex (Irvine, CA, USA).

Techniques: