phosphorylated foxo1 foxo3a  (Cell Signaling Technology Inc)

Journal: Preventive Nutrition and Food Science

Article Title: Standardized Fingerroot ( Boesenbergia pandurata ) Extract Decelerates the Development of Sarcopenia in Aged Rats

doi: 10.3746/pnf.2025.30.1.47

Figure Lengend Snippet: Effects of Boesenbergia pandurata extract (BPE) on protein-degradation-related factors. (A) MuRF1 and atrogin-1 mRNA expression. Values were normalized to the expression of β-actin. (B) p-FoxO3a and FoxO3a protein expression levels. Values were normalized to the expression of the total form of FoxO3a. β-Actin and α-tubulin were used as loading controls. Total RNA and protein were isolated from gastrocnemius muscle tissues. Values are presented as the mean±SD (n=4 per group). # P <0.05, ## P <0.01 (vs. Young group); * P <0.05, ** P <0.01 (vs. Aged group).

Article Snippet: After washing with in Tris-buffered saline containing Tween 20 (TBST), the membranes were incubated with primary antibodies against FoxO3a, phosphorylated (p)-FoxO3a Thr32, PI3K, p-PI3K Tyr458, Akt, p-Akt Ser473, mTOR, p-mTOR Ser2448, p70S6K, p-p70S6K Thr389, 4EBP1, p-4EBP1 Thr37/46, and α-tubulin (1:1,000 dilution; Cell Signaling) overnight at 4°C.

Techniques: Expressing, Isolation

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 3-(4-Hydroxy-3-methoxyphenyl) propionic acid mitigates dexamethasone-induced muscle atrophy by attenuating Atrogin-1 and MuRF-1 expression in mouse C2C12 skeletal myotubes

doi: 10.3164/jcbn.23-70

Figure Lengend Snippet: HMPA suppressed Dex-induced FoxO3a expression. Myotubes were treated as discussed above, followed by the measurement of total and phosphorylated FoxO3a by Western blotting. Western blot, with the protein levels of (A) Total FoxO3a and (B) phosphorylated FoxO3a. The results are expressed as means ± SEM ( n = 3 per group). Dex, dexamethasone; HMPA, 3-(4-hydroxy-3-methoxyphenyl) propionic acid; D+HMPA, Dex + HMPA; P-FoxO3a, phosphorylated FoxO3a. * p <0.05, ** p <0.01, *** p <0.001 indicates a significant difference between indicated groups. MMSTD, molecular mass standard.

Article Snippet: Antibodies against fast- or slow-type myosin heavy chain (MyHC), β-actin (Sigma-Aldrich), total FoxO3a, phosphorylated-FoxO3a, and rabbit IgG (Cell Signaling Technology, Danvers, MA) were used.

Techniques: Expressing, Western Blot

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: 3-(4-Hydroxy-3-methoxyphenyl) propionic acid mitigates dexamethasone-induced muscle atrophy by attenuating Atrogin-1 and MuRF-1 expression in mouse C2C12 skeletal myotubes

doi: 10.3164/jcbn.23-70

Figure Lengend Snippet: Knockdown of KLF15 and FoxO3a diminished protective effects of HMPA. Myotubes were transfected with GR, FoxO3a and NT siRNA as described in the Materials and Methods section, followed by the analysis of (A) GR, (B) KLF15 and (C) FoxO3a mRNA expression. The results are expressed as means ± SEM ( n = 4 per group). * p <0.05, ** p <0.01, *** p <0.001 indicates the significant difference between indicated groups. (D, E) Ubiquitin ligases Atrogin-1 and MuRF-1 expression in NT, GR/KLF15 and FoxO3a-siRNA transfected myotubes. Transfected myotubes were treated with HMPA for 1 ‍h as pretreatment followed by Dex for 24 ‍h. The results are expressed as means ± SEM ( n = 4 per group). * p <0.05, ** p <0.01, *** p <0.001 indicates the significant difference between indicated groups. siRNA, small interfering non-targeting RNA; GR, Glucocorticoid receptor; NT, non-targeting.

Article Snippet: Antibodies against fast- or slow-type myosin heavy chain (MyHC), β-actin (Sigma-Aldrich), total FoxO3a, phosphorylated-FoxO3a, and rabbit IgG (Cell Signaling Technology, Danvers, MA) were used.

Techniques: Knockdown, Transfection, Expressing, Ubiquitin Proteomics

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: Primer sequences used for a quantitative real-time polymerase chain reaction.

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Sequencing

Journal: Cells

Article Title: PAK4 Is Involved in the Stabilization of PD-L1 and the Resistance to Doxorubicin in Osteosarcoma and Predicts the Survival of Diagnosed Patients

doi: 10.3390/cells13171444

Figure Lengend Snippet: The effects of PAK4 expression on proliferation and invasiveness in osteosarcoma cells. ( a , b ) MTT proliferation assays ( a ) and colony-forming assay ( b ) after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. Colony-forming assays were performed after knockdown or overexpression of PAK4 in osteosarcoma cells. U2OS (3 × 10 3 ) and KHOS/NP (3 × 10 3 ) cells were seeded in culture plates for seven days. Western blot for PAK4 and GAPDH was performed to show knockdown and overexpression of PAK4 in osteosarcoma cells. ( c , d ) Migration ( c ) and invasion ( d ) assay after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells. The migration assay was performed by seeding 5 × 10 4 U2OS and 5 × 10 4 KHOS/NP cells in the upper chamber for 48 h. The invasion assay was performed by seeding 1 × 10 5 U2OS and 1 × 10 5 KHOS/NP cells in the upper chamber for 48 h. ( e ) Western blotting for PAK4, FOXO3, phosphorylated FOXO3 (pFOXO3), cyclin D1, P27, BAX, BCL2, snail, TGF-β1, MMP2, and MMP9 after knockdown or overexpression of PAK4 in U2OS and KHOS/NP osteosarcoma cells 24 h after transfection. ( f ) Quantitative reverse-transcription polymerase chain reaction for PAK4, FOXO3, cyclin D1, P27, BAX, BCL2, snail, TGF-β1, MMP2, and MMP9 after knockdown or overexpression of PAK4 in osteosarcoma cells. ( g ) Gross and histologic findings of resected tumors grown in BALB/c nude mice by implanting 1 × 10 6 KHOS/NP cells that were transfected with empty vectors, shRNA for PAK4 , or plasmid for wild-type PAK4 into the marrow space of the right proximal tibia. The tumor volume was measured every seven days with the length × width × height × 0.52 mm 3 equation. The mice were euthanized six weeks after tumor implantation. Resected tumors were H&E stained. ( h ) Gross and histologic findings of pulmonary metastatic nodules in BALB/c nude mice. Arrows indicate metastatic nodules. * p < 0.05; ** p < 0.001; EVs, empty vectors; PAK4-OE, vector for wild-type PAK4 ; shControl, control vector for shRNA; shPAK4, vector for shRNA for PAK4 .

Article Snippet: The following primary antibodies were used in this study: PAK4 (#sc-390507, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PD-L1 (#13684, Cell Signaling Technology, Beverly, MA, USA), cleaved PARP1 (#5625, Cell Signaling Technology, Beverly, MA, USA), cleaved caspase 3 (#9661, Cell Signaling Technology, Beverly, MA, USA), cyclin D1 (#2922, Cell Signaling Technology, Beverly, MA, USA), P27 (#sc-528, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BCL2 (#sc-509, Santa Cruz Biotechnology, Santa Cruz, CA, USA), BAX (#sc-20076, Santa Cruz Biotechnology, Santa Cruz, CA, USA), snail (#sc-271977, Santa Cruz Biotechnology, Santa Cruz, CA, USA/#ab180714, Abcam, Cambridge, UK), transforming growth factor β1 (TGF-β1) (#3709, Cell Signaling Technology, Beverly, MA, USA), MMP2 (#sc-13595, Santa Cruz Biotechnology, Santa Cruz, CA, USA), MMP9 (#sc-21733, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FOXO3 (#2880, Cell Signaling Technology, Beverly, MA, USA), phosphorylated FOXO3 (#9464, Cell Signaling Technology, Beverly, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (#2118, Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Knockdown, Over Expression, Western Blot, Migration, Invasion Assay, Transfection, Reverse Transcription, Polymerase Chain Reaction, shRNA, Plasmid Preparation, Tumor Implantation, Staining, Control

Journal: PLoS ONE

Article Title: Bcl2 Deficiency Activates FoxO through Akt Inactivation and Accelerates Osteoblast Differentiation

doi: 10.1371/journal.pone.0086629

Figure Lengend Snippet: (A–C) Induction of osteoblast differentiation by FoxO3aTM. Primary osteoblasts from calvariae of wild-type mice were infected with adenovirus expressing GFP or FoxO3aTM, and ALP staining at 2 days and von Kossa staining at 6 days after infection (A), quantification of mineralization (B), and osteoblast marker gene expression (C) are shown. The value in GFP-introduced cells was set as 1 and the relative level is shown in B. Similar results were obtained in three independent experiments and representative data are shown. (D–F) Inhibition of the mineralization of MC3T3-E1 cells by sh FoxO1 and sh FoxO3a . MC3T3-E1 cells were infected with retrovirus expressing GFP, sh FoxO1 , or sh FoxO3a , and cultured in the presence of BMP2 (100ng/ml). The expression of FoxO1 and FoxO3a was examined by real-time RT-PCR (D) and mineralization was examined by von Kossa staining (E) and its quantification (F) after culture for 2 weeks. The value in shGFP-introduced cells was set as 1 and the relative levels are shown in F. Similar results were obtained in three independent experiments and representative data are shown.

Article Snippet: Western blot analysis was performed using the following antibodies: anti-Akt, anti-phosphorylated Akt, anti-FoxO1, anti-FoxO3a, anti-phosphorylated FoxO1 (Thr24)/FoxO3a (Thr32), anti-JNK, anti-phosphorylated JNK, anti-Mst1, and anti-phosphorylated Mst1 antibodies (Cell Signaling, Danvers, MA); anti-phosphorylated FoxO3a (S207) antibody (Invitrogen, Tokyo, Japan); and anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Infection, Expressing, Staining, Marker, Inhibition, Cell Culture, Quantitative RT-PCR