Journal: Translational Cancer Research

Article Title: Aspirin inhibits proliferation and metastasis of canine mammary gland tumor cells through Wnt signaling axis

doi: 10.21037/tcr-20-3172

Figure Lengend Snippet: Aspirin inhibits the canonical Wnt/β-catenin pathway in CMGTs. Western blotting showed that Wnt3a, MMP-2, MMP-9, VEGF, p-GSK-3β, PPAR-δ and c-myc were decreased while β-catenin and E-cadherin were increased in CHMp (A) and CHMm (B) cells treated with high concentrations of aspirin (2.5, 5, or 10 mM). Results were expressed as mean ± SD of three independent experiments (n=3). ***, P<0.001 when compared to untreated group.

Article Snippet: Then, 0.1% Triton-X100 1 mL at room temperature was added to these cells for 15 minutes, and incubated with antibodies (β-catenin, p-GSK3β from Bioss, Beijing, China) for 12 h at 4 °C.

Techniques: Western Blot

Journal: Translational Cancer Research

Article Title: Aspirin inhibits proliferation and metastasis of canine mammary gland tumor cells through Wnt signaling axis

doi: 10.21037/tcr-20-3172

Figure Lengend Snippet: Aspirin inhibited the expression and nuclear translocation of β-catenin and p-GSK-3β in CMGTs. CHMp (A) and CHMm (B) cells were seeded onto glass for 15 h and then treated with varying concentrations of Aspirin for 36 h. β-catenin and p-GSK-3β (red) localization was visualized by immunofluorescence followed by confocal imaging. DAPI was used to stain the nuclear (blue). Images are representative of three-independent experiments. Results were expressed as mean ± SD of three independent experiments (n=3).

Article Snippet: Then, 0.1% Triton-X100 1 mL at room temperature was added to these cells for 15 minutes, and incubated with antibodies (β-catenin, p-GSK3β from Bioss, Beijing, China) for 12 h at 4 °C.

Techniques: Expressing, Translocation Assay, Immunofluorescence, Imaging, Staining

Journal: Translational Cancer Research

Article Title: Aspirin inhibits proliferation and metastasis of canine mammary gland tumor cells through Wnt signaling axis

doi: 10.21037/tcr-20-3172

Figure Lengend Snippet: Wnt3a attenuated aspirin-induced inhibition of Wnt/β-catenin signaling and migration and invasion of CMGTs. Wound healing (A,B,C) and Transwell invasion assays (B,C,D,E,F) showed that treatment with Wnt3a (0.1 µg/mL) promoted migration and invasion of CMGTs compared to the untreated group or treatment with only aspirin (5 mM) (0.1% crystal violet staining, magnification, 200×). (G,H,I) Wnt3a elevated p-GSK-3β, β-catenin, MMP-2, MMP-9 and CyclinD1, and it decreased E-cadherin. Results were expressed as mean ± SD of three independent experiments (n=3). *, P<0.05; **, P<0.01; ***, P<0.001 when compared to untreated group; ### , P<0.001 compared to Aspirin group.

Article Snippet: Then, 0.1% Triton-X100 1 mL at room temperature was added to these cells for 15 minutes, and incubated with antibodies (β-catenin, p-GSK3β from Bioss, Beijing, China) for 12 h at 4 °C.

Techniques: Inhibition, Migration, Staining

Journal: Translational Cancer Research

Article Title: Aspirin inhibits proliferation and metastasis of canine mammary gland tumor cells through Wnt signaling axis

doi: 10.21037/tcr-20-3172

Figure Lengend Snippet: FH535 inhibits cell migration, and invasion in CMGTs. (A,B) Wound healing (A) and Transwell invasion assay (B) showed that treatment with the combination of FH535 and Aspirin inhibited migration and invasion of CMGTs compared with untreated group, but as the same effect of treatment with only aspirin (5 mM) or only FH535 (10 uM) (0.1% crystal violet staining, magnification, 200×). (C,D) FH535 elevated E-cadherin, and it decreased p-GSK-3β, β-catenin, MMP-2, MMP-9 and CyclinD1.There was no significant difference between combined drug use and single drug use. Results were expressed as mean ± SD of three independent experiments (n=3). ***, P<0.001 when compared to untreated group.

Article Snippet: Then, 0.1% Triton-X100 1 mL at room temperature was added to these cells for 15 minutes, and incubated with antibodies (β-catenin, p-GSK3β from Bioss, Beijing, China) for 12 h at 4 °C.

Techniques: Migration, Transwell Invasion Assay, Staining

Journal: Antioxidants

Article Title: Blue Light Alters the Composition of the Jejunal Microbiota and Promotes the Development of the Small Intestine by Reducing Oxidative Stress

doi: 10.3390/antiox11020274

Figure Lengend Snippet: Effects of different monochromatic lights on chick jejunum cyclinD1 protein ( A ), Bax protein ( B ), Bcl-2 protein ( C ), Bax/Bcl-2 ratio ( D ), Caspase-3 protein ( E ) p-AKT/total-AKT ratio ( F ), p-GSK-3β/total-GSK-3β ratio ( G ), and β-catenin protein level ( H ) in the jejunums of WL, RL, GL and BL at P42. WL: white light; RL: red light; GL: green light; BL: blue light. These results are shown as means ± SEM. Differences between four groups are presented in the form of different letters ( p < 0.05).

Article Snippet: Next, we used 5% skimmed milk to blocked nitrocellulose membranes for 1 h. Subsequently, the primary antibodies, including anti-Claudin-1 (rabbit, 1:1000; Invitrogen, Carlsbad, CA, USA), anti-Occludin (rabbit, 1:1000;, Invitrogen, Carlsbad, CA, USA), anti-ZO-1 (rabbit, 1:1000; Invitrogen, Carlsbad, CA, USA), anti-phospho-PI3 Kinase (Tyr458) antibody (rabbit, 1:1000, CST, Boston, MA, USA), anti-phosphor-AKT (Ser473) antibody (rabbit, 1:500, CST, Boston, MA, USA), anti-AKT antibody (rabbit, 1:500, CST, Boston, MA, USA), anti-phosphor-GSK-3β antibody (rabbit, 1:500, Abnova, Taiwan, China), anti-GSK-3β antibody (rabbit, 1: 500, Proteintech Group, Inc, Wuhan, China), anti-β-catenin antibody (rabbit, 1:1000, Proteintech Group, Inc, Wuhan, China), anti-cyclinD1 antibody (mouse, 1:200, Abbexa, Cambridge, UK), anti-Bax antibody (goat, 1:1000, Biorbyt, NJ, USA), anti-Bcl-2 antibody (rabbit, 1:1000, Biorbyt, NJ, USA), anti-Caspase-3 antibody (rabbit, 1:1000, CST, Boston, MA, USA) and anti-β-actin (mouse, 1:4000; Co Win Biotech Co., Inc, Beijing, China), were incubated with the membranes overnight at 4 °C.

Techniques:

Journal: Antioxidants

Article Title: Blue Light Alters the Composition of the Jejunal Microbiota and Promotes the Development of the Small Intestine by Reducing Oxidative Stress

doi: 10.3390/antiox11020274

Figure Lengend Snippet: Effects of PI3K inhibitor, AKT inhibitor, GSK-3β inhibitor, GPR43 agonist, GPR41 agonist, Gi inhibitor and Gq inhibitor on p-PI3K protein expression ( A ), p-AKT/total-AKT ratio ( B ), p-GSK-3β/total-GSK-3β ratio ( C ), β-catenin protein expression ( D ), cyclin D1 protein expression ( E ) and CIECs proliferation stimulation index ( F ), Bax protein expression ( G ), Bcl-2 protein expression ( H ) and Caspase-3 protein expression ( I ). LY294002 is an inhibitor of PI3K; GSK-690693 is an inhibitor of AKT; CHIR-99021 is an inhibitor of GSK-3β; 4-CMTB is an agonist of GSK-3β; AR420626 is an agonist of GPR41; PTX is an inhibitor of Gi and Ym254890 is an inhibitor of Gq. Different letters show significant differences among the various treatment groups ( p < 0.05). p-PI3K: phosphorylated PI3-kinase; p-AKT: phosphorylated AKT; p-GSK-3β: phosphorylated GSK-3β. CIECs: chick small intestinal epithelial cells.

Article Snippet: Next, we used 5% skimmed milk to blocked nitrocellulose membranes for 1 h. Subsequently, the primary antibodies, including anti-Claudin-1 (rabbit, 1:1000; Invitrogen, Carlsbad, CA, USA), anti-Occludin (rabbit, 1:1000;, Invitrogen, Carlsbad, CA, USA), anti-ZO-1 (rabbit, 1:1000; Invitrogen, Carlsbad, CA, USA), anti-phospho-PI3 Kinase (Tyr458) antibody (rabbit, 1:1000, CST, Boston, MA, USA), anti-phosphor-AKT (Ser473) antibody (rabbit, 1:500, CST, Boston, MA, USA), anti-AKT antibody (rabbit, 1:500, CST, Boston, MA, USA), anti-phosphor-GSK-3β antibody (rabbit, 1:500, Abnova, Taiwan, China), anti-GSK-3β antibody (rabbit, 1: 500, Proteintech Group, Inc, Wuhan, China), anti-β-catenin antibody (rabbit, 1:1000, Proteintech Group, Inc, Wuhan, China), anti-cyclinD1 antibody (mouse, 1:200, Abbexa, Cambridge, UK), anti-Bax antibody (goat, 1:1000, Biorbyt, NJ, USA), anti-Bcl-2 antibody (rabbit, 1:1000, Biorbyt, NJ, USA), anti-Caspase-3 antibody (rabbit, 1:1000, CST, Boston, MA, USA) and anti-β-actin (mouse, 1:4000; Co Win Biotech Co., Inc, Beijing, China), were incubated with the membranes overnight at 4 °C.

Techniques: Expressing

Journal: Stem Cell Research & Therapy

Article Title: m 6 A-mediated stabilization of Bhlhe22 suppresses osteogenesis in osteoporosis by activating the PI3K-AKT-GSK3β signaling axis

doi: 10.1186/s13287-026-04935-4

Figure Lengend Snippet: Bhlhe22 regulates osteogenesis in BMSCs via PI3K-AKT-GSK3β signaling. A Bubble plot showing KEGG pathway enrichment of DEGs in Bhlhe22 knockdown BMSCs ( n = 3). B GSEA enrichment plots showing downregulation of PI3K-Akt signaling in Bhlhe22 knockdown BMSCs. C Western blot analysis of total and phosphorylated PI3K, AKT, and GSK3β proteins in BMSCs treated with the PI3K activator recilisib (10 µM). From left to right, the mean differences [95% CIs] were: PI3K: -0.7076 [-1.243 to -0.1726], and -0.8059 [-1.341 to -0.2710]; AKT: -0.4206 [-0.8344 to -0.006791], and -0.5912 [-1.005 to -0.1774]; GSK3β: 0.5124 [0.04457 to 0.9803], and 0.6118 [0.1440 to 1.080]. D qPCR analysis of Runx2 , Osx , and Opn mRNA expression in BMSCs subjected to osteogenic induction after treatment as described in (C). From left to right, the mean differences [95% CIs] were: Runx2 : 2.028 [1.745 to 2.311], and 0.5026 [0.2194 to 0.7859]; Osx : 3.195 [2.404 to 3.986], and 0.8793 [0.08827 to 1.670]; Opn : 0.7086 [0.1794 to 1.238], and 0.5397 [0.01050 to 1.069]. E Western blot analysis of RUNX2, OSX, and OPN protein levels in BMSCs treated as described in (C). From left to right, the mean differences [95% CIs] were: RUNX2: 0.8139 [0.2871 to 1.341], and 0.8638 [0.3370 to 1.391]; OSX: 0.9471 [0.5092 to 1.385], and 0.9138 [0.4759 to 1.352]; OPN: 0.4446 [0.01206 to 0.8772], and 0.5913 [0.1588 to 1.024]. F ALP and ARS staining of BMSCs fixed after treatment as described in (C) (scale bar: 200 μm). From left to right, the mean differences [95% CIs] were: ALP: 9.727 [6.678 to 12.78], and 7.394 [4.345 to 10.44]; ARS: 3.171 [2.522 to 3.820], and 1.92 [1.271 to 2.569]. The data represent the means ± SDs ( n = 3). This experiment employed three OP rats, with the BMSCs isolated from each rat being allocated to the siRNA- Bhlhe22 -NC group, siRNA- Bhlhe22 group, and siRNA- Bhlhe22 + Recilisib group. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The full-length blots are presented in the Additional file. Abbreviations: KEGG: Kyoto Encyclopedia of Genes and Genomes; GSEA, gene set enrichment analysis; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; GSK3β, glycogen synthase kinase 3 beta

Article Snippet: p-GSK3β , Abmart , WB , 1:1000.

Techniques: Knockdown, Western Blot, Expressing, Staining, Isolation