Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Relieving colitis by miR-223 through the promotion of macrophage M2 polarization via the modulation of PPAR-γ/FOXO1 signaling. (A) Representative images of western blots indicating the protein expression levels of PPAR-γ and FOXO1. The data represent the findings from three independent experiments. (B, C) Changes in the expression of PPAR-γ and FOXO1. (D, E) Changes in the mRNA levels of PPAR-γ and FOXO1. All the data are expressed as the means ± SDs (n=6 each group). *p<0.05, **p<0.01, ***p<0.001. (F, G) Correlations of colonic miR-223 expression with PPAR-γ and FOXO1 expression in the colon. Pearson analyses were used to correlate miR-223 with colonic PPAR-γ and FOXO1 expression. PPAR-γ, peroxisome proliferator-activated receptor gamma; FOXO1, Forkhead box transcription factor O1.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Immunofluorescence analysis of FOXO1 and M1 macrophage marker CD86 in colonic tissue. Representative micrographs depict protein expression levels of FOXO1 (green) and its co-localization (yellow) with CD86 (red) in colon sections. Nuclei were counterstained with DAPI (blue). Scale bar: 100 µm.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques: Immunofluorescence, Marker, Expressing

Journal: Frontiers in Immunology

Article Title: miR-223 alleviates DSS-induced colitis by prompting macrophage M2 polarization through PPAR-γ/FOXO1 signaling

doi: 10.3389/fimmu.2025.1598781

Figure Lengend Snippet: Schematic representation of the potential mechanisms underlying the targeted therapy of miR-223 supplement for DSS-induced colitis. miR-223 ameliorates DSS-induced colitis through promoting macrophage M2 polarization via modulation of PPAR-γ and FOXO1 signaling. DSS, dextran sodium sulfate.

Article Snippet: After being blocked with bovine serum albumin blocking buffer (3% in PBS), the slides were incubated with the following primary antibodies: anti-PPAR-γ antibody (1:3000) (Cat.# GB11163, Servicebio, Wuhai, China), anti-FOXO1 antibody (1:10000) (Cat.# GB11286-1, Servicebio), anti-CD206 (1:5000) (Cat.# GB113497 , Servicebio), anti-CD86 (1:5000) (Cat.# GB115630 , Servicebio) or anti-F4/80 (1:2000) (Cat.# GB113373 , Servicebio) overnight at 4°C.

Techniques:

Journal: Journal of Orthopaedic Translation

Article Title: FOXO1-mTOR pathway in vascular pericyte regulates the formation of type H vessels to control bone metabolism

doi: 10.1016/j.jot.2024.08.010

Figure Lengend Snippet: Figure 3. FOXO1 inhibition in pericytes prevents angiogenesis. a-c. Representative images (a) and quantitative analyses (b and c) of tube-forming assays of HUVECs. HUVECs were cultured in conditional medium (CM) from HBVPs treated with various with concentrations of AS1842856 (0, 0.1, 1 μM), and collected after 8, 12 and 20 h. Scale bars represent 200 μm. n = 4/group. *P < 0.05, **P < 0.01, ***P < 0.001. d, e. Representative images (d) and wound healing rates (healed area/scratch area%) (e) in the scratch assay showing HUVECs migration under different conditional cultures. Scale bars represent 500 μm. n = 4/group. *P < 0.05, **P < 0.01, ***P < 0.001. f, g. qPCR analysis of VEGF (f) and ANG-1 (g) mRNA expression in HBVPs treated with various concentrations of AS1842856 for 48 h. n = 4/group. *P < 0.05. Data are represented as mean ± SEM and individual points represent independent wells of cultured cells. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test (b, c and f) or Dunnett’s T3 test (e and g).

Article Snippet: To investigate the role of FOXO1 in bone metabolism, we crossed Col2-CreERT mice (RRID: IMSR_JAX: 006774) with Foxo1flox/+mice (Quote: TOS191231MG2-B, Stock No. 017986, Cyagen Bioscience Inc. China) to generate Col2-CreERT/Foxo1flox/flox mice.

Techniques: Inhibition, Cell Culture, Wound Healing Assay, Migration, Expressing

Journal: Journal of Orthopaedic Translation

Article Title: FOXO1-mTOR pathway in vascular pericyte regulates the formation of type H vessels to control bone metabolism

doi: 10.1016/j.jot.2024.08.010

Figure Lengend Snippet: Figure 7. FOXO1 in PDGFRαþ pericytes maintains type H vessels by inhibiting mTOR signaling in mice bone. a. Representative immunofluorescence images of PDGFRα (green) and p-mTOR (red) in the femoral metaphysis of 4-week-old Adipo-Cre/Foxo1f/f and Foxo1f/f mice. Nuclei were stained DAPI. Scale bars represent 50 μm. b, c. Quantitative analysis of PDGFRα+ fluorescence intensity (b) and PDGFRα+ p-mTOR+ colocalization fluorescence intensity (c). n = 6/group. ***P < 0.001. d. Representative immunofluorescence images of PDGFRα (red) and p-mTOR (green) in 8- and 12-month-old mice treated with AS1842856. Nuclei were stained DAPI. Scale bars represent 50 μm. e-g. Quantitative analysis of PDGFRα+ (e), phosphorylated mTOR+ (f), and their colocalization (g) fluorescence signal intensities. n = 6/group. *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Statistical comparisons were made using Student’s t-test (equal variances) (b and c) or one-way ANOVA with Tukey’s post-hoc test (equal variances) (e, f and g).

Article Snippet: To investigate the role of FOXO1 in bone metabolism, we crossed Col2-CreERT mice (RRID: IMSR_JAX: 006774) with Foxo1flox/+mice (Quote: TOS191231MG2-B, Stock No. 017986, Cyagen Bioscience Inc. China) to generate Col2-CreERT/Foxo1flox/flox mice.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A Volcano plot of differentially expressed genes after combination treatment. B KEGG pathway enrichment highlighting apoptosis, cell-cycle, and FoxO signaling pathways. C GSEA showing enrichment of a FoxO1-related gene set (NES = 1.39812, p = 0.00244). D Heatmap of genes within the PI3K pathway (e.g., CDKN1A , CDKN1B , FoxO1 , FoxO3 , FoxO4 ). E – G Western blots for PI3K-p110δ, AKT, p-AKT, PPARα, and FoxO family proteins in Karpas-422, RL, and Sc-1 cells; the combination suppressed PI3K/AKT signaling and increased PPARα and FoxO1 (representative of three independent experiments). H Subcellular fractionation with Western blot showing increased nuclear FoxO1 after combination treatment (representative of three independent experiments). I Densitometric analysis of the nuclear/cytoplasmic ratio of FoxO1 ( n = 3). J qPCR showing increased FoxO1 mRNA after dual treatment ( n = 3). K Immunofluorescence images demonstrating increased FoxO1 expression and nuclear localization following combination treatment. Scale bars as indicated.

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Protein-Protein interactions, Western Blot, Fractionation, Immunofluorescence, Expressing

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A Volcano plot of differential metabolites in RL cells (Chiglitazar vs control: 88 upregulated, 333 downregulated). B KEGG enrichment of altered metabolites highlighting FoxO signaling, carbon metabolism, and central carbon metabolism in cancer. C Integrated KEGG analysis combining transcriptomic and metabolomic data, identifying the FoxO pathway among the top-enriched pathways. D – F Functional assays showing reduced glucose uptake, lactate production, and pyruvate levels after Chiglitazar ( n = 3). G Western blots showing downregulation of HIF-1α, GLUT1, PGK1, p-AKT, and phosphorylated FoxO1 (T24, S256) (representative of three independent experiments).

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Control, Metabolomic, Functional Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A qPCR confirming FoxO1 mRNA knockdown in RL cells stably expressing sh FoxO1 #1 or sh FoxO1 #2 ( n = 3). B Western blot validation of FoxO1 protein reduction (representative of three independent experiments). C Densitometric analysis showing relative FoxO1 protein levels (~50% for sh FoxO1 #1; ~37% for sh FoxO1 #2; n = 3). D CCK-8 assays showing reduced drug sensitivity in FoxO1-knockdown cells treated with Linperlisib and Chiglitazar ( n = 3). E Annexin V/PI apoptosis assays showing decreased apoptosis upon combination treatment in FoxO1-deficient cells ( n = 3). F EdU assays showing increased DNA synthesis in knockdown cells under drug treatment ( n = 3). G Western blots of FoxO1 downstream targets (Bim, Bax, p21, p27) in control and FoxO1-knockdown cells (representative of three independent experiments).

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Knockdown, Stable Transfection, Expressing, Western Blot, Biomarker Discovery, CCK-8 Assay, DNA Synthesis, Control

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A PDX study design. B Representative tumor images at endpoint. C Tumor weights at endpoint ( n = 6 per group). D Tumor volume curves over time ( n = 6 per group). E Mouse body-weight curves; a mild reduction was observed in the Linperlisib group ( n = 6 per group). F Ki-67 IHC and G quantification ( n = 6 per group). Scale bars as indicated. H p-AKT IHC and I quantification showing reduced PI3K pathway activation in the combination group ( n = 6 per group). Scale bars as indicated. J FoxO1 IHC and K quantification showing enhanced nuclear FoxO1 in the combination group ( n = 6 per group). Scale bars as indicated.

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: Activation Assay

Journal: Clinical Cancer Research

Article Title: Forkhead Transcription Factor FOXO1 is a Direct Target of Progestin to Inhibit Endometrial Epithelial Cell Growth

doi: 10.1158/1078-0432.ccr-10-1287

Figure Lengend Snippet: Figure 4. Induction of FOXO1 by progestin. A, EM-E6/E7/TERT/PR cells preincubated in growth media for 24 h were incubated in the serum-depleted phenol red–free DMEM in the absence or presence of MPA or progesterone for 48 h at the indicated concentrations. Nuclear or cytoplasmic extracts were collected from cells and the western blot analysis was performed using FOXO1 antibody. Lamin and b–actin were used as controls for nuclear or cytoplasmic protein loading, respectively. B, immunocytochemistry of FOXO1. EM-E6/E7/TERT/PR cells preincubated in growth media for 24 h were incubated on LAB TEK chamber slides for 24 h in the absence or presence of MPA for 24 h. After fixation, the cells were incubated with primary antibody to FOXO1, followed by fluorescent anti-IgG secondary antibody. The cells were also incubated with DAPI for nuclear staining and observed under a fluorescence microscope. Note that FOXO1 expression is induced preferentially in the nuclei (consistent with DAP staining) by MPA treatment. D, mouse tumors formed with EM-E6/E7/TERT/RAS/PR cells were treated with or without progesterone (P4) via subcutaneous injection of hormone pellets and were collected 4 weeks after the treatment. Then, whole-cell extracts were prepared, followed by western blot analysis for FOXO1. Immunohistochemistry of FOXO1 was also performed with matched samples of the EM-E6/E7/TERT/RAS/PR tumors treated with or without P4. HE staining of the tumor samples and induced FOXO1 expression mainly in the nuclei of the tumor cells are shown.

Article Snippet: EM-E6/E7/TERT/PR cells were cultured on LAB TEK chamber slides (Nalge Nunc International) for 24 h and treated with or without MPA (10 nM) for 24 h. Then, the cells were fixed with 10% formaldehyde neutral buffer solution (37152-51; Nacalai Tesque, Inc.), immersed in methanol for 10 min at 20 C, blocked with PBS containing 10% goat serum and 0.3% Triton X-100 (166-11805; Wako Pure Chemical Industries, Ltd.) for 1 h at room temperature, and stained with monoclonal antibody to FOXO1 (#9462; Cell Signaling Technology) at 1:100 dilution for 12 h at 4 C. Next, they were incubated with fluorescent anti-rabbit IgG conjugates [Alexa Fluor 568 goat anti-rabbit IgG (HþL) highly cross-adsorbed, A11036; Invitrogen] for 1 h at room temperature in the dark.

Techniques: Incubation, Western Blot, Immunocytochemistry, Staining, Fluorescence, Microscopy, Expressing, Injection, Immunohistochemistry

Journal: Clinical Cancer Research

Article Title: Forkhead Transcription Factor FOXO1 is a Direct Target of Progestin to Inhibit Endometrial Epithelial Cell Growth

doi: 10.1158/1078-0432.ccr-10-1287

Figure Lengend Snippet: Figure 5. Mechanisms of FOXO induction by progestin. A, RT-PCR analysis of FOXO1 in EM-E6/E7/TERT/PR or EM-E6/E7/TERT cells. The cells were cultured with or without MPA in the presence or absence of cycloheximide (CHX) for different time periods and RNAs were collected and subjected to the RT-PCR for FOXO1. B, luciferase reporter assay using FOXO promoter. EM-E6/E7/TERT or EM-E6/E7/TERT/PR cells were transfected with luciferase reporter plasmid containing 2.0 kb of FOXO promoter or with blank reporter plasmid (pGL3-basic), followed by treatment with or without MPA. Seventy-two hours after treatment, cells were recovered and luciferase assays were performed. Relative luciferase activity is shown in each reporter plasmid. Data are presented as mean SD of the three independent experiments. *P < 0.05. C, EM-E6/E7/TERT/PR cells were treated with or without MPA at 10 nM and incubated at different time periods, followed by the western blot analysis for PTEN or p-AKT.

Article Snippet: EM-E6/E7/TERT/PR cells were cultured on LAB TEK chamber slides (Nalge Nunc International) for 24 h and treated with or without MPA (10 nM) for 24 h. Then, the cells were fixed with 10% formaldehyde neutral buffer solution (37152-51; Nacalai Tesque, Inc.), immersed in methanol for 10 min at 20 C, blocked with PBS containing 10% goat serum and 0.3% Triton X-100 (166-11805; Wako Pure Chemical Industries, Ltd.) for 1 h at room temperature, and stained with monoclonal antibody to FOXO1 (#9462; Cell Signaling Technology) at 1:100 dilution for 12 h at 4 C. Next, they were incubated with fluorescent anti-rabbit IgG conjugates [Alexa Fluor 568 goat anti-rabbit IgG (HþL) highly cross-adsorbed, A11036; Invitrogen] for 1 h at room temperature in the dark.

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Incubation, Western Blot

Journal: Clinical Cancer Research

Article Title: Forkhead Transcription Factor FOXO1 is a Direct Target of Progestin to Inhibit Endometrial Epithelial Cell Growth

doi: 10.1158/1078-0432.ccr-10-1287

Figure Lengend Snippet: Figure 6. Role of FOXO1 in the action of progestin. EM-E6/E7/TERT/PR cells were transfected with non-specific scramble siRNAcontrol or FOXO1- specific siRNA. Forty-eight hours after transfection, the cells were incubated with or without MPA at 10 nM for 72 h. Then, western blot analysis was performed to confirm the levels of FOXO1 expression. Simultaneously, cell number was counted in paired samples and shown as the relative value (proliferation index) in each sample to evaluate the effects of MPA. Data are presented as mean SD of the three independent experiments. *P < 0.05.

Article Snippet: EM-E6/E7/TERT/PR cells were cultured on LAB TEK chamber slides (Nalge Nunc International) for 24 h and treated with or without MPA (10 nM) for 24 h. Then, the cells were fixed with 10% formaldehyde neutral buffer solution (37152-51; Nacalai Tesque, Inc.), immersed in methanol for 10 min at 20 C, blocked with PBS containing 10% goat serum and 0.3% Triton X-100 (166-11805; Wako Pure Chemical Industries, Ltd.) for 1 h at room temperature, and stained with monoclonal antibody to FOXO1 (#9462; Cell Signaling Technology) at 1:100 dilution for 12 h at 4 C. Next, they were incubated with fluorescent anti-rabbit IgG conjugates [Alexa Fluor 568 goat anti-rabbit IgG (HþL) highly cross-adsorbed, A11036; Invitrogen] for 1 h at room temperature in the dark.

Techniques: Transfection, Incubation, Western Blot, Expressing

Journal: Clinical Cancer Research

Article Title: Forkhead Transcription Factor FOXO1 is a Direct Target of Progestin to Inhibit Endometrial Epithelial Cell Growth

doi: 10.1158/1078-0432.ccr-10-1287

Figure Lengend Snippet: Figure 7. Akt activity limits the MPA effect. A, EM-E6/E7/TERT/ PR/DN-AKT or EM-E6/E7/TERT/ PR/vector cells were established from EM-E6/E7/TERT/PR cells by retroviral transfection of dominant negative Akt gene or blank vector, respectively. These cells were treated with or without MPA for different periods, and the cell number was counted to evaluate the proliferative activity. Data are presented as mean SD of the three independent experiments. B, western blot analysis was performed using nuclear extracts of EM-E6/E7/TERT/PR/DN-AKT or EM-E6/E7/TERT/PR/vector cells in the absence or presence of MPA on day 3 or day 6 to compare the levels of nuclear FOXO1 induction. Data are presented as mean SD of the three independent experiments.

Article Snippet: EM-E6/E7/TERT/PR cells were cultured on LAB TEK chamber slides (Nalge Nunc International) for 24 h and treated with or without MPA (10 nM) for 24 h. Then, the cells were fixed with 10% formaldehyde neutral buffer solution (37152-51; Nacalai Tesque, Inc.), immersed in methanol for 10 min at 20 C, blocked with PBS containing 10% goat serum and 0.3% Triton X-100 (166-11805; Wako Pure Chemical Industries, Ltd.) for 1 h at room temperature, and stained with monoclonal antibody to FOXO1 (#9462; Cell Signaling Technology) at 1:100 dilution for 12 h at 4 C. Next, they were incubated with fluorescent anti-rabbit IgG conjugates [Alexa Fluor 568 goat anti-rabbit IgG (HþL) highly cross-adsorbed, A11036; Invitrogen] for 1 h at room temperature in the dark.

Techniques: Activity Assay, Plasmid Preparation, Retroviral, Transfection, Dominant Negative Mutation, Western Blot

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 1. Rapid depletion of SOX2 and OCT4 affects the accessibility landscape of thousands of sites.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 2. Loss of SOX2 and OCT4 effect accessibility at shared and independent regions.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques:

Journal: The EMBO journal

Article Title: Pioneer activity distinguishes activating from non-activating SOX2 binding sites.

doi: 10.15252/embj.2022113150

Figure Lengend Snippet: Figure 5. Open chromatin regions maintained by SOX2 are associated with transcription.

Article Snippet: For the knock-in of the FKBP sequence at the genes of interest, we used previously described plasmids and approach (SOX2 fkbpdonor, Addgene # 175552; SOX2 sgRNA, Addgene #175553; NANOG donor, Addgene # 175554; NANOG sgRNA, Addgene # 175555) Briefly, cells were transfected with the plasmids containing the gRNA sequence and the donor plasmid designed to include the FKBP-2xHA-P2A-[GFP/mCherry] in between two homology arms for the gene of interest.

Techniques: