Journal: Cell reports

Article Title: Combined inhibition of de novo glutathione and nucleotide biosynthesis is synthetically lethal in glioblastoma

doi: 10.1016/j.celrep.2025.115596

Figure Lengend Snippet: (A–E) TERT mRNA (A), ATRX mRNA (B), GSH pool size (C), percentage of 13 C labeling of GSH from [U- 13 C]-glutamine (D), and GCLC mRNA (E) in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (F) Western blots for GCLC in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. β-actin was used as the loading control. (G) GCL activity in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (H) Western blots for phosphorylated and total FOXO1 in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (I) FOXO1 binding to the GCLC promoter as measured by ChIP-qPCR in NHA CONTROL , NHA TERT , and NHA ATRX-KO cells. (J) Western blots for the FLAG tag in NHA CONTROL , NHA TERT , and NHA TERT cells expressing a FLAG-tagged constitutively active form of FOXO1 (CA-FOXO1). β-actin was used as the loading control. (K) FOXO1 binding to the GCLC promoter measured by ChIP-qPCR in NHA CONTROL , NHA TERT , and NHA TERT cells expressing CA-FOXO1. (L–N) GCLC protein expression (L), GSH pool size (M), and percentage of 13 C labeling of GSH from [U- 13 C]-glutamine (N) in NHA CONTROL , NHA TERT , and NHA TERT cells expressing a FLAG-tagged CA-FOXO1. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns indicates lack of statistical significance. See also .

Article Snippet: Human FOXO1 primers , Origene , HP205770.

Techniques: Labeling, Control, Western Blot, Activity Assay, Binding Assay, ChIP-qPCR, FLAG-tag, Expressing, Standard Deviation

Journal: Cell reports

Article Title: Combined inhibition of de novo glutathione and nucleotide biosynthesis is synthetically lethal in glioblastoma

doi: 10.1016/j.celrep.2025.115596

Figure Lengend Snippet: (A–C) FOXO1 binding to the GCLC promoter (A), GCLC protein expression (B), and GSH pool size (C) in GBM6, U251, SB28, SF10417, A375, and HepG2 cells transfected with non-targeting control small interfering RNA (siRNA; siNT) or two non-overlapping siRNA sequences against TERT (siTERT-1 and siTERT-2). (D–F) FOXO1 binding to the GCLC promoter (D), GCLC mRNA (E), and GSH pool size (F) in BT142, KNS42, and U2OS cells transfected with an empty vector or with a plasmid expressing ATRX. (G–I) TERT mRNA (G), FOXO1 binding to the GCLC promoter (H), and GCLC mRNA (I) in GBM, astrocytoma, or gliosis biopsies. Data are presented as mean ± standard deviation. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns indicates lack of statistical significance.

Article Snippet: Human FOXO1 primers , Origene , HP205770.

Techniques: Binding Assay, Expressing, Transfection, Control, Small Interfering RNA, Plasmid Preparation, Standard Deviation

Journal: PloS one

Article Title: The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1.

doi: 10.1371/journal.pone.0080807

Figure Lengend Snippet: Figure 2. Binding of miR-96 in the 3’ UTR of FOXO1. LNCaP cells were transfected with 10 nM pre-miR-96, anti-miR-96, pre- miR-NC #1 or combination of pre-miR-96 and anti-miR-96 as well as 500 ng pMiR report β-Galactosidase control plasmid and 500 ng pMiR report plasmid for FOXO1 3’ UTR A) binding site 1 at position 264-270 and B) binding site 2 at position 2138-2145. * P <0.05, **P <0.01, One-way ANOVA and Tukey’s multiple comparison post test.

Article Snippet: Transfection of PCa cell lines with pre-miRNAs, miRNA inhibitors and plasmid DNA LNCaP and DU145 cells were transfected with pre-miRNA precursors, anti-miRNA inhibitors or pre-miR-NC#1 (Applied Biosystems) in a final concentration of 10 nM or 500 ng FOXO1 full length clone (Origene, Rockville MD, USA) using siPort NeoFX transfection agent (Applied Biosystems).

Techniques: Binding Assay, Transfection, Control, Plasmid Preparation, Comparison

Journal: PloS one

Article Title: The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1.

doi: 10.1371/journal.pone.0080807

Figure Lengend Snippet: Figure 3. Effect of miR-96 overexpression on FOXO1 expression. Prostate cancer cells were transfected with 10 nM pre- miR-96, anti-miR-96, pre-miR-NC #1 or combination of pre-miR-96 and anti-miR-96. miR-96 expression in (A) LNCaP and (B) DU145 cells and FOXO1 expression in (C) LNCaP and (D) DU145 cells. Data are shown as mean (+SD) of three independent assays. ** P <0.01, *** P <0.001, One-way ANOVA. FOXO1, AKT, and pAKT protein expression in (E) LNCaP and (F) DU145 cells was visualized by western blotting. β-Actin was used as a loading control.

Article Snippet: Transfection of PCa cell lines with pre-miRNAs, miRNA inhibitors and plasmid DNA LNCaP and DU145 cells were transfected with pre-miRNA precursors, anti-miRNA inhibitors or pre-miR-NC#1 (Applied Biosystems) in a final concentration of 10 nM or 500 ng FOXO1 full length clone (Origene, Rockville MD, USA) using siPort NeoFX transfection agent (Applied Biosystems).

Techniques: Over Expression, Expressing, Transfection, Western Blot, Control

Journal: PloS one

Article Title: The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1.

doi: 10.1371/journal.pone.0080807

Figure Lengend Snippet: Figure 4. FOXO1 overexpression in prostate cancer cell lines. LNCaP and DU145 cells were transfected with 500 ng FOXO1 full length clone or empty vector, 10 nM pre-miR-96 or pre-miR-NC #1. FOXO1 expression in (A) LNCaP and (B) DU145 cells. Data are shown as mean (+SD) of three independent assays. *** P <0.001, One-way ANOVA. FOXO1 protein expression in (C) LNCaP and (D) DU145 cells was visualized by western blotting. β-Actin was used as a loading control. Apoptosis in CPT-treated (E) LNCaP and (H) DU145 cells. 24 h after transfection cells were treated with 10 µM CPT for 24 h. Cells were stained with Annexin V-FITC and PI. Fraction of early (black bars) and late (white bars) apoptotic cells was measured by flow cytometry. Data are shown as mean (+SD) of three independent assays. ns, P>0.05,*P <0.05,**P<0.01,***P<0.001, Two-way ANOVA, Bonferroni’s post test). Cell cycle transition was measured by PI staining in transfected (F) LNCaP and (I) DU145 cells. Cells were serum starved one day after transfection for another 24 hrs and subsequently fixed and stained. Black, G1-phase; light grey, S-phase; dark grey, G2/M-phase. Data are shown as mean of three independent assays. ***P<0.001, One-way ANOVA. Analysis of subG1 peak in (G) LNCaP and (J) DU145 cells. Data are shown as mean of three independent assays. ***P<0.001, One-way ANOVA.

Article Snippet: Transfection of PCa cell lines with pre-miRNAs, miRNA inhibitors and plasmid DNA LNCaP and DU145 cells were transfected with pre-miRNA precursors, anti-miRNA inhibitors or pre-miR-NC#1 (Applied Biosystems) in a final concentration of 10 nM or 500 ng FOXO1 full length clone (Origene, Rockville MD, USA) using siPort NeoFX transfection agent (Applied Biosystems).

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Staining, Flow Cytometry

Journal: PloS one

Article Title: The antiapoptotic function of miR-96 in prostate cancer by inhibition of FOXO1.

doi: 10.1371/journal.pone.0080807

Figure Lengend Snippet: Figure 5. miR-96 expression and FOXO1 expression in PCa specimens. (A) miR-96 and FOXO1 mRNA expression was measured by RT-qPCR in 69 matched PCa (PC) and normal adjacent tissue (PN). All data were normalized to efficiency and interplate control. FOXO1 expression was normalized to the reference gene TUBA1B [19] and miR-96 expression was normalized to miR-130b [6]. Data are shown as median expression (+ interquartile range). FOXO1 expression is displayed on the left y-axis, miR-96 expression on the right y-axis. ***P <0.001; Wilcoxon signed rank test. (B) Haematoxilin-eosin (H/E) staining was performed to identify tumor ducts. (C+D) FOXO1 expression was detected by immunhistochemistry on a tissue microarray containing tissue cores corresponding to 69 PCa specimen. Each core had a diameter of 1.5 mm.

Article Snippet: Transfection of PCa cell lines with pre-miRNAs, miRNA inhibitors and plasmid DNA LNCaP and DU145 cells were transfected with pre-miRNA precursors, anti-miRNA inhibitors or pre-miR-NC#1 (Applied Biosystems) in a final concentration of 10 nM or 500 ng FOXO1 full length clone (Origene, Rockville MD, USA) using siPort NeoFX transfection agent (Applied Biosystems).

Techniques: Expressing, Quantitative RT-PCR, Control, Staining, Microarray

Journal: PloS one

Article Title: NLK is a novel therapeutic target for PTEN deficient tumour cells.

doi: 10.1371/journal.pone.0047249

Figure Lengend Snippet: Figure 3. PTEN/NLK synthetic lethality is abrogated by FOXO1 silencing. (A) Survival analysis of HCT116 cells transfected with NLK and/or FOXO1 siRNA. HCT116 PTEN2/2 and HCT116 PTEN+/+ cells were transfected with siRNA targeting NLK and FOXO1 as shown and surviving fractions determined after five days. The p value (*) was calculated using Student’s t test. (B) Nuclear localisation of FOXO1 is enhanced in PTEN deficient cells upon NLK silencing. HCT116 isogenic cells were co-transfected with GFP-tagged FOXO1 cDNA in addition to control (non targeting) siRNA (siCON) or NLK siRNA. Two days later cells were fixed and stained with DAPI. Green signal represents GFP-FOXO1 and blue signal represents nuclear DAPI (nuclear) staining. Arrows indicate cells with nuclear localisation of FOXO1. (C) Senescence is increased by NLK siRNA in PTEN deficient cells. Bar chart of relative relative senescence levels caused by NLK silencing are shown. HCT116-derived PTEN isogenic cell lines were reversed transfected with a pool of two validated siRNAs against NLK, as well as siCON pool#2 (Dharmacon) as non-targeting control, using RNAiMAX (Invitrogen). Seven days after transfection cells were fixed and incubated overnight at 37uC in a solution containing X-gal. (D) Representative images for b-Galactosidase staining of PTEN deficient cells. Blue staining indicates b-Galactosidase. doi:10.1371/journal.pone.0047249.g003

Article Snippet: FOXO1 detection HCT116 isogenic cells were co-transfected with GFP-tagged FOXO1 cDNA (Origene) in addition to control (non targeting) siRNA (siCON) or NLK siRNA.

Techniques: Transfection, Control, Staining, Derivative Assay, Incubation

Journal: FEBS open bio

Article Title: FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells.

doi: 10.1002/2211-5463.13696

Figure Lengend Snippet: Fig. 1. FOXO1 inhibition with AS1842856 induced a set of Hallmark WNT beta-catenin genes based on GSEA. (A) GSEA was performed with RNA-Seq data upon 1 lM AS1842856 treatment for 48 h in BT549 cells. Enrichment plot of Hallmark WNT and beta-catenin. The profile of the running enrichment score (ES) and rank-ordered list are shown for GSEA enrichment. (B) Heatmap of 27 core-enriched genes in HALLMARK_WNT_BETA_CATENIN signaling gene set.

Article Snippet: The FOXO1 gene was disrupted in U87MG cells using the OriGene CRISPR Cas9 knock-out kit Cat#: KN400477 (Rockville, MD).

Techniques: Inhibition, RNA Sequencing

Journal: FEBS open bio

Article Title: FOXO1 promotes the expression of canonical WNT target genes in examined basal-like breast and glioblastoma multiforme cancer cells.

doi: 10.1002/2211-5463.13696

Figure Lengend Snippet: Fig. 4. FOXO1 RNAi-targeting or disruption impacted WNT target gene expression. (A) FOXO1 and/or beta-catenin (CTNNB1) RNAi led to reduced LEF1, TCF7, and GADD45A gene expression 24 h post-transfection in U87MG cells by qRT-PCR using TUBB as the control. (B) FOXO1 RNAi led to reduced LEF, and GADD45A gene expression 18 h post-transfection in BT549 cells by qRT-PCR using TUBB as the con- trol. (C, D) FOXO1 disruption by CRISPR Cas9 genome editing in U87MG cells led to reduced LEF1 and TCF7 gene expression measured by qRT-PCR using TUBB as the control; disruption of FOXO1 produced a truncated protein as assessed by western blot analysis. The disrup- tion mutant is predicted to express the C-terminal portion of FOXO1 starting in the middle of the DNA-binding domain. (E) Exogenous FOXO1-H215R (DNA-binding-defective) significantly restored TCF7 gene expression to FOXO1 disruption mutants. * denotes significantly dif- ferent by Student’s t-Test compared with the control (P < 0.05) with SD error bars. Each experiment had three biologically independent replicates.

Article Snippet: The FOXO1 gene was disrupted in U87MG cells using the OriGene CRISPR Cas9 knock-out kit Cat#: KN400477 (Rockville, MD).

Techniques: Disruption, Targeted Gene Expression, Gene Expression, Transfection, Quantitative RT-PCR, Control, CRISPR, Produced, Western Blot, Mutagenesis, Binding Assay

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: RT-qPCR primer sequences.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques:

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: FoxO1 primer sequences.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques:

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: FoxO1 expression was downregulated while SREBP-1c and fatty acid metabolism genes were upregulated in HB. (a) The relative expression levels of FoxO1, SREBP-1c, FASN, ACLY, ACC, and MAGL were detected by RT-qPCR. (b) WB was used to measure the expressions of FASN, ACLY, ACC, and MAGL. (c) IHC evaluated the expression levels of FASN. (d) RT-qPCR was performed to examine the relative expressions of FoxO1, SREBP-1c, and FASN. The magnification is 100 or 400 times, and the corresponding scale bar is 100 μ m or 25 μ m; & P < 0.05 vs. the control group in (a–c); & P < 0.05 vs. the WRL68 group in (d). All experiments were performed 5 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: FoxO1 deficiency led to upregulation of SREBP-1c expression and enhanced proliferation, migration, invasion of HB. (a) RT-qPCR detected the relative expression levels of FoxO1, SREBP-1c, and FASN. (b) CCK8 assay was used to detect cell proliferation. (c) Cell migration was evaluated by scratch assay. (d) Transwell assay was performed to examine cell invasion. The magnification is 100 times, scale bar = 100 μ m; & P < 0.05 vs. the oe-NC group; # P < 0.05 vs. the si-NC group. All experiments were performed 3 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: Expressing, Migration, Quantitative RT-PCR, CCK-8 Assay, Wound Healing Assay, Transwell Assay

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: FoxO1 deficiency enhanced fatty acid metabolism in HB. (a) ATP Assay Kit was used to detect the concentration of total cellular ATP. (b) Human FAO ELISA Kit was adopted to examine FAO. (c) TG Assay Kit detected TG. (d) NEFA was examined with Nonesterified Free Fatty Acids Assay Kit. (e) Cellular Acetyl-CoA level was evaluated with Human Acetyl-CoA ELISA Kit. & P < 0.05 vs. the oe-NC group; # P < 0.05 vs. the si-NC group. All experiments were performed 3 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: ATP Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: FoxO1 and SREBP-1c inhibited each other and regulated Huh-6 cell functions. (a) RT-qPCR detected the relative expression levels of FoxO1 and SREBP-1c. (b) ChIP was performed to detect the direct interaction between the FoxO1 gene and SREBP-1c protein. (c) CCK8 assay was used to detect cell proliferation. (d) Cell migration was evaluated by scratch assay. (e) Transwell assay was performed to examine cell invasion. The magnification is 100 times, scale bar = 100 μ m; & P < 0.05 vs. the si-NC group, ∗ P < 0.05 vs. the si-FoxO1 group, and # P < 0.05 vs. the si-SREBP-1c group. All experiments were performed 3 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Migration, Wound Healing Assay, Transwell Assay

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: Coordinated regulation of FoxO1 and SREBP-1c regulated fatty acid in Huh-6 cells. (a) ATP Assay Kit was used to detect the concentration of total cellular ATP. (b) Human FAO ELISA Kit was adopted to examine FAO. (c) TG Assay Kit detected TG. (d) NEFA was examined with Nonesterified Free Fatty Acids Assay Kit. (e) Cellular Acetyl-CoA level was evaluated with Human Acetyl-CoA ELISA Kit. & P < 0.05 vs. the si-NC group. All experiments were performed 3 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: ATP Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: Coordinated regulation of FoxO1 and SREBP-1c facilitated the progression of HB in vivo. (a) Photograph of subcutaneous neoplasia in nude mice. (b) Volume and weight of tumor tissues. (c) The relative expression levels of FoxO1 and SREBP-1c were detected by RT-qPCR. (d) HE staining was performed to observe tumor tissues. The magnification is 100 or 400 times, and the corresponding scale bar is 100 μ m or 25 μ m; & P < 0.05 vs. the si-NC group. All experiments were performed 3 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Staining

Journal: Mediators of Inflammation

Article Title: The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism

doi: 10.1155/2021/5754592

Figure Lengend Snippet: Coordinated regulation of FoxO1 and SREBP-1c regulated fatty acid metabolism in vivo. (a) The concentration of FASN was detected by IF. (b) The relative expression levels of FASN, ACLY, ACC, and MAGL were evaluated by RT-qPCR. (c) WB was used to measure the expressions of FASN, ACLY, ACC, and MAGL. The magnification is 400 times, scale bar = 25 μ m; & P < 0.05 vs. the si-NC group. All experiments were performed 3 times.

Article Snippet: The si-FoxO1 and si-SREBP-1c vectors were obtained by integrating the shRNA sequence targeting human FoxO1 or SREBP-1c into a psi-LVRU6MP lentivirus vector (GeneCopoeia).

Techniques: In Vivo, Concentration Assay, Expressing, Quantitative RT-PCR