Journal: Scientific reports

Article Title: LncRNA HOXA-AS3 promotes cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma.

doi: 10.1038/s41598-024-67596-4

Figure Lengend Snippet: Figure 3. LncRNA HOXA-AS3 interacts with miR-218-5p in osteosarcoma cells. (A) Potential binding sites between miR-218-5p and lncRNA HOXA-AS3 and FOXP1. (B) RT-PCR data showed that miR-218-5p inhibitors decreased the expression of miR-218-5p. In addition, miR-218-5p mimic transfection increased the miR-218-5p expression levels. (C) Dual luciferase reporter assays showed that miR-218-5p interacted with lncRNA HOXA-AS3. miR-218-5p regulated luciferase activity in HOXA-AS3 wild-type group. (D) Dual luciferase reporter assays showed that miR-218-5p interacted with and regulated FOXP1. **p < 0.01 versus control group. (E) Western blotting analysis data showed that lncRNA HOXA-AS3 and miR-218-5p regulate the expression of FOXP1 in osteosarcoma cells. WT: wild type; MUT: mutant.

Article Snippet: The primary antibody FOXP1 (1:2000, #2005, Cell Signaling Technology) were used to incubate the membrane at cold room overnight.

Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Luciferase, Activity Assay, Control, Western Blot, Mutagenesis

Journal: Scientific reports

Article Title: LncRNA HOXA-AS3 promotes cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma.

doi: 10.1038/s41598-024-67596-4

Figure Lengend Snippet: Figure 4. Downregulation of lncRNA HOXA-AS3 reduces colony formation via miR-218-5p and FOXP1. (A) Colony formation assays were performed to measure the colony formation in osteosarcoma cells co-transfected with sh-HOXA-AS3 and pcDNA FOXP1, or sh-HOXA-AS3 and miR-218 inhibitors. (B) Quantitative data are shown for colony formation. ***p < 0.001 versus control group. #p < 0.05; ##p < 0.01 versus sh-HOXA-AS3 alone.

Article Snippet: The primary antibody FOXP1 (1:2000, #2005, Cell Signaling Technology) were used to incubate the membrane at cold room overnight.

Techniques: Transfection, Control

Journal: Scientific reports

Article Title: LncRNA HOXA-AS3 promotes cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma.

doi: 10.1038/s41598-024-67596-4

Figure Lengend Snippet: Figure 5. Downregulation of lncRNA HOXA-AS3 reduces cell migration via miR-218-5p and FOXP1. (A) Wound healing assays were performed to measure migratory ability in osteosarcoma cells co-transfected with sh-HOXA-AS3 and pcDNA FOXP1, or sh-HOXA-AS3 and miR-218 inhibitors. (B) Quantitative data are shown for colony formation. **p < 0.01; ***p < 0.001 versus control group. #p < 0.05; ##p < 0.01 versus sh-HOXA-AS3 alone.

Article Snippet: The primary antibody FOXP1 (1:2000, #2005, Cell Signaling Technology) were used to incubate the membrane at cold room overnight.

Techniques: Migration, Transfection, Control

Journal: Scientific reports

Article Title: LncRNA HOXA-AS3 promotes cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma.

doi: 10.1038/s41598-024-67596-4

Figure Lengend Snippet: Figure 6. Downregulation of lncRNA HOXA-AS3 reduces cell invasion via miR-218-5p and FOXP1. (A) Transwell assays were performed to measure cell invasive ability in osteosarcoma cells co-transfected with sh-HOXA-AS3 and pcDNA FOXP1, or sh-HOXA-AS3 and miR-218 inhibitors. (B) Quantitative data are shown for colony formation. ***p < 0.001 versus control group. #p < 0.05; ##p < 0.01 versus sh-HOXA-AS3 alone.

Article Snippet: The primary antibody FOXP1 (1:2000, #2005, Cell Signaling Technology) were used to incubate the membrane at cold room overnight.

Techniques: Transfection, Control

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) pAAV-ChR2-mCherry virus was injected into the cortex of one hemisphere at P1, acute slices were prepared at P14–18, and SPNs were recorded in the contralateral striatum (created with BioRender.com ). ( B ) Average (± SEM) EPSC responses triggered by blue light stimulation from D1 and D2 SPNs in D2 Foxp1 CTL (blue and black, left) and in D2 Foxp1 cKO mice (green and red, right). ( C ) EPSC amplitudes in D1/D2 SPN pairs in D2 Foxp1 CTL and D2 Foxp1 cKO mice. ( D ) D2/D1 ratio of EPSC amplitude for each D2/D1 pair in (C). ( E ) Latency to EPSC onset in D2 SPNs. ( F ) Time taken by the EPSC response to reach the peak amplitude in D2 SPNs. ( G ) Average (± SEM) EPSCs during a 5-pulse train of blue light in D2 SPNs. ( H ) Ratio of second to fifth response amplitude with respect to (wrt) first response amplitude during a stimulus train in D2 SPNs. ( I ) Example traces showing mEPSCs in D2 SPNs. ( J ) mEPSC frequency (left) and amplitude (right) of D2 SPNs in both D2 Foxp1 CTL and D2 Foxp1 cKO mice. Ns in all figures are (D2 Foxp1 CTL ; D2 Foxp1 cKO ). For (B) to (D), 20 D1/D2 pairs in five mice and 19 D1/D2 pairs in four mice. For (E) and (F), 20 D2 SPNs in five mice and 19 D2 SPNs in four mice. For (G) and (H), 18 D2 SPNs in five mice and 14 D2 SPNs in four mice. For (J), 10 D2 SPNs in three mice and 15 D2 SPNs in three mice. Statistics: (C) Two-way analysis of variance (ANOVA) with Fisher’s LSD test; (D) Mann-Whitney (MW) test; (E, F, and J) t test; and (H) Mixed effects model with Holm-Sidak corrections for multiple comparisons. ** P < 0.01, *** P < 0.001, and **** P < 0.001.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques: Virus, Injection, MANN-WHITNEY

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Monosynaptic EPSCs were evoked by depolarization of presynaptic terminals during blockade of voltage-dependent Na + channels. Average EPSC responses (±SEM) from D1 and D2 SPNs in slices from D2 Foxp1 CTL mice (blue and black, respectively, left) and from D2 Foxp1 cKO mice (green and red, respectively, right). AMPAR-mediated EPSC amplitude is measured as a response peak at −70-mV holding potential, whereas for NMDAR-mediated EPSC amplitude, it is the average response in a 90- to 100-ms window while holding at +50 mV (highlighted with a brown box). ( B ) Amplitude of AMPAR-mediated EPSCs for each pair of D1/D2 SPNs in D2 Foxp1 CTL and D2 Foxp1 cKO mice (left). D2/D1 ratio of the EPSC amplitude for each pair (right). ( C ) Amplitude of NMDAR-mediated EPSCs and D2/D1 EPSC amplitude ratio. ( D and E ) Ratio of AMPAR- to NMDAR-mediated EPSC amplitudes for D2 and D1 SPNs in D2 Foxp1 CTL and D2 Foxp1 cKO mice. ( F ) Ratio of second-fifth response amplitude with respect to first response during a stimulus train in D2 SPNs. Ns in all figures are (D2 Foxp1 CTL ; D2 Foxp1 cKO ). For (A) to (C), 16 D1/D2 pairs in five mice and 16 D1/D2 pairs in three mice. For (D), 21 D2 SPNs in five mice and 18 D2 SPNs in three mice. For (E), 17 D1 SPNs in five mice and 18 D1 SPNs in three mice. For (F), 18 D2 SPNs in five mice and 14 D2 SPNs in three mice. Statistics: [B (left) and C (left)], two-way ANOVA with Fisher’s LSD test; [B (right) and C (right)] MW test; (D and E) t test; and (F) mixed effects model with Holm-Sidak correction for multiple comparisons. ** P < 0.01, *** P < 0.001, and **** P < 0.001.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques:

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Average (±SEM) EPSC responses from D1 and D2 SPNs in D1 Foxp1 CTL mice (blue and black, respectively, left) and from D1 Foxp1 cKO mice (green and red, respectively, right). ( B ) Amplitude of AMPAR-mediated EPSCs for D1/D2 SPN pairs in D1 Foxp1 CTL and D1 Foxp1 cKO mice (interaction term, genotype × cell type; P < 0.01). ( C ) D1/D2 ratio of EPSC amplitude for each D1/D2 pair. ( D ) Example traces of sEPSC in D1 SPNs. ( E ) Frequency (left) and amplitude (right) of sEPSCs in D1 SPNs in D1 Foxp1 CTL mice and D1 Foxp1 cKO mice. Ns in all figures are (D1 Foxp1 CTL ; D1 Foxp1 cKO ). For (A) to (C), 21 D1/D2 pairs in three mice and 21 D1/D2 pairs in four mice). For (E), 17 D1 SPNs in three mice and 21 D1 SPNs in four mice. Statistics: (B) Two-way ANOVA with Fisher’s LSD test, (C) MW test, and (E) t test.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques:

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Schematic of approach: blue light of different intensities was applied where subthreshold stimuli generated EPSPs, while threshold or suprathreshold stimuli generated action potentials. ( B ) Excitability curves from D1/D2 SPN pairs in D2 Foxp1 CTL (left) and D2 Foxp1 cKO mice (right). ( C ) Fraction of SPNs with at least one action potential evoked at the maximum possible light intensity stimulus. A total of 15% of D2 SPNs in D2 Foxp1 CTL mice and 50% in D2 Foxp1 cKO mice did not fire action potentials. ( D ) D2/D1 EPSC amplitude ratio for each D1/D2 pair at −70 mV. ( E ) D2/D1 ratio of EPSP amplitude for each D1/D2 pair. Ns in all figures are (D2 Foxp1 CTL ; D2 Foxp1 cKO ). For (B) and (C), 20 D1/D2 pairs in six mice and 26 D1/D2 pairs in seven mice. For (D), 15 D1/D2 pairs in three mice and 9 D1/D2 pairs in two mice. For (E), 14 D1/D2 pairs in three mice and 7 D1/D2 pairs in two mice. Statistics: (B) Mixed effects model with Sidak correction for multiple comparisons, (C) two-way ANOVA with Fisher’s LSD test, and (D and E) t test. ** P < 0.01 and *** P < 0.001.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques: Generated

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Example image: A metal stimulating electrode was placed in underlying cortical white matter to stimulate the corticostriatal projections and responses were recorded in pairs of Foxp1- deleted ( Foxp1 D2_vcKO , eGFP positive) and control ( Foxp1 D2_Flox , eGFP negative) D2 SPNs. ( B ) Average EPSC responses (±SEM) at −70 mV from Foxp1 -deleted (magenta) and control (gray) D2 SPNs. ( C ) EPSC amplitude for the pairs of Foxp1 D2_vcKO and Foxp1 D2_Flox D2 SPNs. ( D ) Example traces of mEPSCs in control and Foxp1- deleted D2 SPNs. ( E ) mEPSC frequency (left) and amplitude (right) for control and Foxp1- deleted D2 SPNs. ( F ) Total charge (frequency × amplitude) transfer in D2 SPNs of both groups. For (B) and (C), 7 pairs of D2 SPNs in four mice. For (E) and (F), 12 Foxp1 D2_Flox and 14 Foxp1 D2_vcKO D2 SPNs from three mice. Statistics: (C) Paired t test, [E (left) and F] MW test, and [E (right) t test]. * P < 0.05 and ** P < 0.01.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques: Control

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Schematic describing Control AAV and Foxp1 AAV virus injection in the striatum and recording from D2 SPNs (created with BioRender.com ). ( B ) eGFP-positive D2 SPNs and mCherry positive Foxp1 AAV -infected neurons in D2 Foxp1 cKO mice. Foxp1 AAV -infected D2 SPNs (merge, yellow) are highlighted with arrows. ( C ) Example traces of action potential firing in D2 SPNs from D2 Foxp1 CTL + Control AAV ; D2 Foxp1 CTL + Foxp1 AAV ; D2 Foxp1 cKO + Control AAV ; D2 Foxp1 cKO + Foxp1 AAV mouse groups. ( D ) Action potential (AP) number versus current step amplitude curves (F-I curves) for these D2 SPNs. ( E ) Input resistance of D2 SPNs at −55, −65, and −85 mV for all four mouse groups. ( F ) Current density versus voltage curves at different voltage steps (−20 to −140 mV). ( G ) Example traces of mEPSCs in D2 SPNs from all four mouse groups. ( H ) mEPSC frequency (left) and mEPSC amplitude (right) of D2 SPNs from all four mouse groups. Ns in all figures are (D2 Foxp1 CTL + Control AAV ; D2 Foxp1 CTL + Foxp1 AAV ; D2 Foxp1 cKO + Control AAV ; D2 Foxp1 cKO + Foxp1 AAV ). For (D) and (E), 36 D2 SPNs in three mice and 34 D2 SPNs in four mice and 33 D2 SPNs in five mice and 32 D2 SPNs in eight mice. For (F), 37 D2 SPNs in three mice and 34 D2 SPNs in three mice and 44 D2 SPNs in four mice and 31 D2 SPNs in three mice. For (H), 36 D2 SPNs in three mice and 31 D2 SPNs in three mice and 40 D2 SPNs in four mice and 28 D2 SPNs in three mice. Statistics: (D to F) Mixed effect analysis with Holm-Sidak correction for multiple comparisons. */**/*** show the statistics between Foxp1 AAV + D2 Foxp1 cKO and Control AAV + D2 Foxp1 cKO D2 SPNs. (H) One-way ANOVA with Holm-Sidak correction for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques: Control, Virus, Injection, Infection

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Schematic showing the experimental strategy for differential gene expression (DGE) analysis of (i) “all” D2 SPNs from D2 Foxp1 CTL mice and D2 Foxp1 cKO mice and (ii) “selected” D2 SPNs from D2 Foxp1 CTL mice infected with Control AAV and D2 SPNs from D2 Foxp1 cKO mice infected with Control AAV and Foxp1 AAV (created with BioRender.com ). ( B ) UMAP for SPNs from the three groups of mice shows distinct clusters of the three SPN types. Different shades of red, green, and blue are indicative of subclusters in D1 SPNs, D2 SPNs, and eSPNs, respectively. ( C ) Volcano plot from DGE analysis of D2 SPNs from D2 Foxp1 CTL and D2 Foxp1 cKO demonstrate 1,538 genes down-regulated and 1222 genes up-regulated with Foxp1 deletion in D2 SPNs. [false discovery rate (FDR) ≤ 0.05, and log 2 fold change ≥ 0.378]. ( D ) Gene Ontology plot for synaptic genes using SynGO for down-regulated DEGs. Gray color represents the subcategories of synaptic genes that do not overlap with DEGs, while blue color indicates a significant overlap (−log 10 FDR > 1.3), where darker blue color implies smaller FDR values. The number of overlapping DEGs is stated below each category. ( E ) Gene Ontology plot for synaptic genes using SynGO for up-regulated DEGs. Gray color corresponds to no overlap, while red color demonstrates significant overlap, where darker red color depicts smaller FDR values. ( F ) Volcano plot from DGE analysis for D2 SPNs from D2 Foxp1 cKO mice infected with Control AAV and Foxp1 AAV show 19 down-regulated and 59 up-regulated genes with Foxp1 reinstatement (FDR ≤ 0.05 and log 2 fold change ≥ 0.378). ( G ) Expression heat map of 78 genes in each sample from three groups ( Foxp1 CTL + Control AAV , Foxp1 cKO + Control AAV , and Foxp1 cKO + Foxp1 AAV ). Gene symbols in red and blue shades are up-regulated and down-regulated DEGs, respectively, in D2 SPNs with Foxp1 reinstatement. FC, fold change.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques: Gene Expression, Infection, Control, Expressing

Journal: Science Advances

Article Title: FOXP1 regulates the development of excitatory synaptic inputs onto striatal neurons and induces phenotypic reversal with reinstatement

doi: 10.1126/sciadv.adm7039

Figure Lengend Snippet: ( A ) Latency to fall from the rotarod in four consecutive trials for each of the 3 days (total 12 trials) in Foxp1 CTL + Control AAV , Foxp1 CTL + Foxp1 AAV , D2 Foxp1 cKO + Control AAV , and D2 Foxp1 cKO + Foxp1 AAV mice. ( B and C ) Velocity and time spent in the periphery of the open-field box for Foxp1 CTL + Control AAV , Foxp1 CTL + Foxp1 AAV , D1D2 Foxp1 cKO + Control AAV , and D1D2 Foxp1 cKO + Foxp1 AAV mice. ( D ) Nesting score of the mice from all four groups as stated in (B) and (C). We used the same control mice ( Foxp1 CTL ) for the comparison with D2 Foxp1 cKO and D1D2 Foxp1 cKO mice groups. Number of mice were 27 Foxp1 CTL + Control AAV , 21 Foxp1 CTL + Foxp1 AAV , 20 D2 Foxp1 cKO + Control AAV , 15 D2 Foxp1 cKO + Foxp1 AAV , 10 D1D2 Foxp1 cKO + Control AAV , and 10 D1D2 Foxp1 cKO + Foxp1 AAV mice. Statistics: (A) Two-way ANOVA with Holm-Sidak correction for multiple comparisons and (B to D) one-way ANOVA with Holm-Sidak correction for multiple comparisons. * P < 0.05, *** P < 0.001, and **** P < 0.0001. ns, not significant.

Article Snippet: The primary antibodies were FOXP1 (in-house), mCherry (Proteintech, catalog no. 26765-1-AP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore, catalog no. MAB374), actin (Millipore, catalog no. MAB1501).

Techniques: Control, Comparison

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A ) RNA collected from E12.5 control and Foxp1 cKO cortices. ( B ) Gene ontology terms associated with significantly misregulated genes in the Foxp1 cKO cortex at E12.5. GO terms: BP, biological process; KEGG, pathways, REAC, reactome pathway. ( C ) Significantly misregulated genes associated with angiogenesis (magenta), glycolysis (blue), and HIF-1α signaling (white/hashed lines) in Foxp1 cKOs at E12.5. * and bars with hashed lines are also HIF-1α targets. ( D – F ) 3D surface rendering of IB4 + blood vessels in the cortex from E12.5 to E14.5 with Foxp1 and TUJ1 expression. White dashed arrows delineate cortical plate. ( G – J ) Foxp1 intensity, periventricular plexus vessel (PVP) number, volume, and cortical plate area in the lateral cortex at E12.5–E14.5. ( K ) Number of Tbr2 + progenitors (per 200 μm 2 ) in wild-type cortex at E12.5–E14.5. Scale bars 100 μm. Data information: Statistical significance determined by ANOVA ( B , C ). N = 4 controls, 2 mutants ( B , C ). N = 5 ( G ), 4 ( H ), 3–4 ( I ), 3–4 ( J ), 3–4 ( K ) embryos/time point. All data represented as mean ± SEM. .

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: Control, Expressing

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A ) Volcano plot of gene expression changes in the absence of Foxp1 in E12.5 lateral cortex compared to control embryos. Gray circles denote non-significant gene changes (adjusted p -value > 0.05); red circles denote significantly differentially expressed genes (adjusted p -value < 0.05). ( B ) Human disorders associated with genes significantly misregulated in Foxp1 cKO mutants at E12.5. ( C ) The principal component analysis (PCA) of control and Foxp1cKO mutants shows PC1 and PC2. ( D – F ) IHC for Tbr2 + intermediate progenitors in wild-type cortex at E12.5, E13.5, and E14.5. Schematic denotes area image in ( D – F ). Scale bars 50 μm. Data information: significance determined by ANOVA ( A , B ).

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: Gene Expression, Control

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A – H ) IHC analysis of HIF-1α and Foxp1 in Nestin + RG at E11.5 and E12.5 wild-type cortex. ( I – L ) IHC analysis of Pax6 and HIF-1α expression in the medial and dorsolateral cortex at E12.5 in control and Foxp1 cKO mutants. Boxed areas indicate regions quantified in ( M ) and ( N ). ( M , N ) HIF-1α mean gray value (percent control) and medial to dorsal ratio in control and Foxp1 cKO mutant cortex at E12.5. ( O ) Quantification of mRNA fold enrichment (normalized to Actb ) for Slc2a1, Ldha, Vegfa, Aldoa, Pfkfb3, Slc16a4, Pfkl, Pdk1 , and Actb in control and Foxp1 cKO by qPCR in the lateral cortex at E12.5. ( P – U ) IHC for Glut1, Ldha, Vegfa, and Nestin in control and Foxp1 cKO mutants in the VZ of the cortex at E12.5. The inset is Glut1/Ldha/Vegfa only. ( V ) Glut1 mean gray value (percent control) in control and Foxp1 cKO mutant Nestin + RG at E12.5. ( W ) Ldha mean gray value (percent control) in control and Foxp1 cKO cortex at E12.5. ( X ) Vegfa mean gray value (percent control) in control and Foxp1 cOn cortex at E12.5. ( Y ) Summary of HIF-1α and target gene expression in control and Foxp1 cKO cortex at E12.5. Dashed lines demarcate the ventricular zone. Scale bars 50 μm ( A – L ), 20 μm ( P – U ). Data information: p = 0.0003 and 0.0055, respectively, Student’s t-test. N = 7 control, 6 mutants ( M , N ). N = 3 control, 3 mutants (3 litters). p values = 0.039 ( Slc2a1 ), 0.0256 ( Ldha ), 0.013 ( Vegfa ), 0.0073 ( Aldoa ), 0.0177 ( Pfkfb3 ), 0.0152 ( Slc16a4 ), 0.0183 ( Pfkl ), 0.0392 ( Pdk1 ). Student’s t-test ( O ). p = 0.0262, Student’s t-test. N = 7 control, 3 mutants ( V ). p = 0.0169, Student’s t-test. N = 5 control, 4 mutants ( W ). p = 0.0328, Students t-test. N = 4 control, 5 mutants ( X ). All data represented as mean ± SEM. .

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: Expressing, Control, Mutagenesis, Targeted Gene Expression

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A – C ) RNA Scope analysis of Vegfa and Foxp1 in the wild-type cortex at E12.5–E14.5. ( D ) Vegfa mean gray value (over background) in the ventricular zone between E12.5–E14.5. ( E – G ) IHC for Vegfa and Nestin in the wild-type cortex at E12.5, E13.5, and E14.5. ( H – K ) High magnification images of Vegfa, Nestin, and IB4 at the apical surface of the VZ at E13.5. ( L – S ) High magnification images of IHC for Vegfa, Nestin, and Foxp1 at E12.5 and E14.5 in wild-type cortex. ( T – U ) IHC for Vegfa in the cortex at E12.5 in control and Foxp1 cKO embryos. Cyan boxes represent areas in CP magnified in T ,’ and U .’ Magenta boxes represent the area within VZ magnified in T ” and U ”. ( V ) Quantification of Vegfa mean gray values in the cortical plate in control and Foxp1 cKO cortex at E12.5. Scale bars 50 μm ( A – G , T , U ) 10 μm ( H – S ). Data information: N = 5–7 embryos per time point ( D ). N = 3 control, 6 mutant (2 litters). p = 0.8674, Student’s t-test ( V ). All data represented as mean ± SEM.

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: RNAscope, Control, Mutagenesis

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A , B ) Isolectin B4 (IB4) vessel staining in the cortex of control and Foxp1 cKO embryos at E12.5. ( C , D ) Surfaced rendered contiguous vessels in ( A ) and ( B ). ( E ) Number of IB4 + vessels in each binned area in control and Foxp1 cKO lateral cortices at E12.5. ( F ) Schematic of bins used for quantification in ( E ). ( G , H ) IB4 labeled filopodia at the ventricular surface in control and Foxp1 cKO cortex at E12.5. Arrowheads mark filopodia. ( I – J ) IB4 + vessels in control and Foxp1 cKO cortex at E13.5. ( K , L ) Surface rendered cortex images in control and Foxp1 cKO at E13.5. ( M ) Mean filopodia (percent control) in control and Foxp1 cKO cortex at E12.5. ( N – P ) Vessel length, vessel volume, and number of branches per vessel in control and Foxp1 cKO cortex. Dashed yellow lines demarcate the apical surface. d, dorsal; v, ventral. Scale bars 100 μm ( A – D ), 5 μm ( G , H ), and 50 μm ( I – L ). Data information: p < 0.0001, Mann–Whitney test. N = 10 control, 7 mutants ( E ). p = 0.0033, Student’s t-test. N = 12 control, 6 mutants ( M ). p = 0.0386, 0.0235, and 0.0063, respectively, Student’s t-test. N = 6 control, 7 mutants ( N – P ). All data represented as mean ± SEM. .

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: Staining, Control, Labeling, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A ) Western blot analysis of Foxp1 and GAPDH protein in control and Foxp1 KO mouse embryonic stem cells. ( B ) Western blot analysis and quantification of HIF-1α protein (compared to α-tubulin) in control and Foxp1 KO spheroids at 10 div treated with DMSO or VH298. ( C ) qPCR analysis of HIF-1α target gene expression in control and Foxp1 KO#1 spheroids at 10 div. ( D ) qPCR analysis of HIF-1α target gene expression in control and Foxp1 KO#1 spheroids at 10 div. treated with DMSO or VH298. ( E – J ) IHC for Pax6 + NPCs and Ctip2 + neurons in control and Foxp1 KO spheroids (10 div) treated with DMSO or VH298. ( K ) Percentage of DAPI + cells that are Pax6 + in control and Foxp1 KO spheroids (10 div) treated with DMSO or VH298. ( L ) Percentage of DAPI + cells that are Ctip2 + in control and Foxp1 KO spheroids (10 div) treated with DMSO or VH298. ( M ) Schematic of control and Foxp1 cKO phenotypes. Foxp1 attenuates the HIF-1α signaling pathway to promote RG self-renewal. In the absence of Foxp1, HIF-1α target gene expression is upregulated, angiogenesis is perturbed, and RG precociously differentiate. Scale bars 100 μm. Data information: p = **0.0097, 0.00364 (Foxp1 KO#1 ) and 0.0162 (Foxp1 KO#2 ). Student’s t-test. N = 10–12 spheroids, western blots run in triplicate ( B ). p values = 0.0008 ( Slc2a1 ), <0.0001 ( Ldha ), 0.0026 ( Vegfa ), 0.0010 ( Aldoa ), <0.0001 ( Pdk1 ), <0.001 ( Pfkl ), 0.0061 ( Pfkfb3 , Mann–Whitney test), 0.0001 ( Slc16a4 ). N = 10–12 spheroids/experiment, 3 experiments ( C ). p values = <0.0001 ( Slc2a1 , control), 0.0013 ( Ldha , control), 0.0446 ( Ldha , Foxp1 KO#1 ), 0.0002 ( Vegfa , control), 0.0179 ( Aldoa , control), 0.0002 ( Pdk1 , control), 0.0201 ( Pkfl , control), 0.0003 ( Slc16a4 , control). N = 10–12 spheroids/experiment, 3 experiments ( D ). One-way ANOVA, p = >0.9999, <0.0001, 0.928, 0.0024 and 0.1066, respectively. N = 10–12 spheroids/experiment, 3 experiments ( K ). Ordinary one-way ANOVA, p = >0.9999, <0.0001, 0.8306, 0.0196, 0.9883. N = 10–12 spheroids/experiment, 3 experiments ( L ). All data represented as mean ± SEM. .

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: Western Blot, Control, Targeted Gene Expression, MANN-WHITNEY

Journal: EMBO Reports

Article Title: Foxp1 suppresses cortical angiogenesis and attenuates HIF-1alpha signaling to promote neural progenitor cell maintenance

doi: 10.1038/s44319-024-00131-8

Figure Lengend Snippet: ( A – C ) IHC for Pax6 in control and Foxp1 KO spheroids at 7 days in vitro (div). ( D – F ) IHC for Ctip2 and Tbr1 + neurons in control and Foxp1 KO spheroids at 7 div. ( G ) qPCR analysis of HIF-1α target gene expression in control and Foxp1 KO#2 spheroids at 10 div. ( H – P ) IHC for Foxp1 in Pax6 + NPCs in control and Foxp1 KO spheroids at 10 div. ( Q ) qPCR analysis of HIF-1α target gene expression in control and Foxp1 KO#2 spheroids at 10 div. treated with DMSO or VH298. Scale bars 100 μm ( A – F ), 50 μm ( H – P ). Data information: N = 10–12 spheroids from 3 individual batches. p = 0.0007 ( Slc2a1 ), 0.0108 ( Ldha ), 0.0031 ( Vegfa ), 0.0009 ( Aldoa ), 0.0002 ( Pdk1 ), 0.0002 ( Pfkl ), 0.0226 ( Pfkfb3 ), <0.0001 ( Slc16a4 ). Student’s t-test ( G ). p = <0.0001 ( Slc2a1 , control), 0.0013 ( Ldha , control) <0.0001 ( Vegfa , control), 0.0179 ( Aldoa , control) 0.0002 ( Pdk1 , control), 0.0312 ( Pdk 1, Foxp1 KO#2 ), 0.0201 ( Pfkl , control), 0.0003 ( Slc16a4 , control). Student’s t-test ( Q ). All data represented as mean ± SEM.

Article Snippet: Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204).

Techniques: Control, In Vitro, Targeted Gene Expression

Journal: The Journal of Clinical Investigation

Article Title: FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

doi: 10.1172/JCI89511

Figure Lengend Snippet: (A) Representative image of IHC analysis. IHC showed overlapping of FOXP1 (green) and nestin (red) in fibroblast-like cells adjacent to endosteal (white arrows) in neonatal BM. Scale bar: 100 μm. (B) Western blotting for the FOXP1 protein levels in BM MSCs. Western blotting for BM MSCs at 1, 8, and 30 months detected 3 major isoforms: FOXP1A (95 kD), FOXP1D (70 kD), and FOXP1C (50 kD). n = 3. (C) qPCR for the relative expression of Foxp1 isoforms AB, ABD, ACD, and ABCD in MPCs from BM of 1 and 8 months old. n = 3. (D) Relative expression levels of FOXP1 in young and aged hMPC as detected by qPCR. (E) Western blotting for FOXP1 protein levels in human BM MSCs from donors ages 26, 27, 33, 41, 74, 75 and 82 years. (F) Inverse expressions of Foxp1 and p16INK4A (p16) were assessed by qPCR in primitive MSCs obtained from BM of 1-, 8-, and 30-month-old mice. (G) Inverse correlation of Foxp1 and p16INK4A expression levels during in vitro expansion and passaging (P1–P6) of murine MSCs. n = 3. (H) Methylation of CpG islands within the Foxp1 promoter variant 3 (–196 to 1) as detected by bisulfite sequencing in 1- and 12-month-old MSCs. Black circles represent methylated CpG islands and white circles unmethylated CpG islands. n = 3. *P < 0.05; **P < 0.01.

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies to mouse FOXP1 (Millipore, ABE68, 1:100) and Nestin (Abcam, ab11306, 1:100) diluted in TBST with 1% BSA.

Techniques: Western Blot, Expressing, In Vitro, Passaging, Methylation, Variant Assay, Methylation Sequencing

Journal: The Journal of Clinical Investigation

Article Title: FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

doi: 10.1172/JCI89511

Figure Lengend Snippet: (A) The osteogenic and adipogenic potential of MSCs from FOXP1 mutant BM was assessed 14 days after induction of differentiation by ALP and oil red O staining. n = 5. (B and C) Quantification of the frequencies of osteoblast clones (CFU-Ob) and adipocyte clones (CFU-Ad) following induction in A. n = 3. (D and E) Expression of bone (Alp, Col1a1) and fat (Pparg, Cebpa, and Fabp4) markers as assessed by quantitative reverse-transcriptase PCR (qRT-PCR) of uninduced MSCs in BM. n = 3. (F) Foxp1 was overexpressed in C3H10T1/2 cells by retrovirus (pMSCV-FOXP1) and cultured in differentiation medium. Cell differentiation was assessed 6 days after adipogenic induction by oil red O staining or 14 days after osteogenic induction by ALP staining. n = 3. Scale bar: 100μm (G) The expression of adipogenic markers (Cebpa, Pparg, and Fabp4) was analyzed 6 days after adipogenic induction of FOXP1-expressing C3H10T1/2 cells. n = 3. (H) The expression of bone markers (Runx2, Alp, Col1a1, and Osterix) was assessed by qPCR 14 days after osteogenic culture of FOXP1-overexpressing C3H10T1/2 cells. n = 3. *P < 0.05; ***P < 0.001.

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies to mouse FOXP1 (Millipore, ABE68, 1:100) and Nestin (Abcam, ab11306, 1:100) diluted in TBST with 1% BSA.

Techniques: Mutagenesis, Staining, Clone Assay, Expressing, Reverse Transcription, Quantitative RT-PCR, Cell Culture, Cell Differentiation

Journal: The Journal of Clinical Investigation

Article Title: FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

doi: 10.1172/JCI89511

Figure Lengend Snippet: (A and B) In vitro co-IP of FOXP1-His with CEBPβ-Flag (A) or CEBPδ-Flag (B) following transfection into 3T3-L1 cells. (C) Western blotting detection of CEBPβ and CEBPδ in FOXP1 immunoprecipitates from primary BM MSCs. (D) Colocalization of FOXP1 and CEBPβ, CEBPδ or RBPjκ in the nuclei of C3H10T1/2 cells transfected with FOXP1-His and CEBPβ/δ- or RBPjκ-expressing vectors. Green, anti-His antibody; red, antibodies for CEBPβ, CEBPδ, or RBPjκ; blue, DAPI staining for nucleus. Scale bar: 50 μm. (E) FOXP1 represses the transactivation ability of CEBPβ and CEBPδ in inducing Pparg-Luc luciferase activity in C3H10T1/2 cells. n = 3. (F) Western blot detection of PPARγ and FABP4 levels in Foxp1Prx1Δ/Δ mutant BM at 8 months. (G) qRT-PCR of Pparg mRNA levels in MSCs. n = 3. (H and I) Promoter occupancy of Pparg gene as assessed by anti-FOXP1 ChIP-PCR in MSCs. (J and K) Co-IP of FOXP1 with RBPjκ in C3H10T1/2 cells (J) and BM MSCs (K). (L) FOXP1 repression of luciferase activity as judged by Rbpjk-Luc reporter activity following the induction by NICD in C3H10T1/2 cells. n = 3. (M and N) qPCR confirms relatively higher expression levels of Hey1 and Heyl in MSCs from Foxp1Prx1Δ/Δ mutant BM (M) or C3H10T1/2 cells transfected with pMSCV-FOXP1 (N). n = 3. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies to mouse FOXP1 (Millipore, ABE68, 1:100) and Nestin (Abcam, ab11306, 1:100) diluted in TBST with 1% BSA.

Techniques: In Vitro, Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, Staining, Luciferase, Activity Assay, Mutagenesis, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

doi: 10.1172/JCI89511

Figure Lengend Snippet: (A) In vitro population doublings of MSCs from Foxp1Prx1Δ/Δ BM at 1 and 6 months. n = 4. (B) FACS analysis dot plot of BrdU+ labeled MSCs (defined as CD31–CD45–CD29+Sca1+) following 48-hour pulse-chase in BM of 3-month-old mice. n = 3. (C) Quantification of the percentages of BrdU+ MSCs in 3-month-old mice. n = 4. (D) qPCR assessment of cell-cycle inhibitor (p16INK4A, p21, and p27) expression in BM MSCs. n = 3. (E) Giemsa staining for the CFU-F colonies of BM MSCs from Foxp1Prx1Δ/Δ mutant mice at 3, 6, and 12 months. n = 3. (F) Quantification of the number of CFU-F colonies in E. n = 3. (G) Western blot detection of FOXP1, p16INK4A, H3K9me3, and LAP2β protein levels in BM MSCs. (H) SA–β-gal staining, Ki67, γH2AX, and LAP2β immunostaining of the second passage of MSCs. Scale bars: 50 μm. (I and J) Quantification of Ki67+ and γH2AX+ frequencies in MSCs from H. n = 3. (K) Quantification of the DCFDA fluorescence intensities reveals increased ROS levels in MSCs (CD31–CD45–CD29+Sca1+) from Foxp1Prx1Δ/Δ BM at 3 months old. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies to mouse FOXP1 (Millipore, ABE68, 1:100) and Nestin (Abcam, ab11306, 1:100) diluted in TBST with 1% BSA.

Techniques: In Vitro, Labeling, Pulse Chase, Expressing, Staining, Mutagenesis, Western Blot, Immunostaining, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

doi: 10.1172/JCI89511

Figure Lengend Snippet: (A) Promoter occupancy of p16INK4A as assessed by anti-FOXP1 ChIP-PCR in MSCs. Sequence analysis identified a consensus FOXP1-binding site within the p16INK4A promoter (1701-1695). (B) FOXP1 repression of p16INK4A transcription as revealed by p16-Luc reporter assays in C3H10T1/2 cells transfected with the indicated levels of cotransfected FOXP1. (C) FOXP1 repressed transcription of p16-Luc reporter, but not the mutant reporter p16-Luc (mut). (D) Doubling times of BM MSCs expanded in vitro. The replication capacity of Foxp1Prx1Δ/Δ p16–/– double mutants is significantly higher than that of Foxp1Prx1Δ/Δ single mutants, but lower than that of control mice. n = 4. (E) Representative images of μCT analyses of trabecular bones of tibia in 3-month single (Foxp1Prx1Δ/Δ) and double (Foxp1Prx1Δ/Δ p16–/–) mutants. (F) Bone volume, BMD, and number of trabecular bones are significantly rescued in Foxp1Prx1Δ/Δ p16–/– double-mutant compared with Foxp1Prx1Δ/Δ single-mutant mice. n = 4. *P < 0.05; **P < 0.01; ***P < 0.001. Tb. BV/TV, trabecular bone volume/total volume; Tb. N, trabecular number; Tb. Sp, trabecular spacing; Tb. Th, trabecular thickness.

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies to mouse FOXP1 (Millipore, ABE68, 1:100) and Nestin (Abcam, ab11306, 1:100) diluted in TBST with 1% BSA.

Techniques: Sequencing, Binding Assay, Transfection, Mutagenesis, In Vitro, Control

Journal: The Journal of Clinical Investigation

Article Title: FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging

doi: 10.1172/JCI89511

Figure Lengend Snippet: (A–D) In vitro expansion of hMPCs isolated from BM donors ages 27 (male, A), 75 (female, B), 82 (female, C), and 74 (male, D) years. MPCs were transfected with lentiviral LV-FOXP1 or LV-GFP. Control 27 yr, hMPCs were from a 27-year-old male donor. (E) Expression of FOXP1 and p16INK4A in the fifth passage of hMPCs with FOXP1 overexpression from a 74-year-old donor. (F) Representative images of ALP and oil red O staining 14 days after osteogenic differentiation of the 74-year-old donor’s hMPCs transfected with LV-FOXP1. Scale bar: 100μm. (G) Western blot for PPARγ and FABP4 expression in adipogenic cultures of hMPCs in F. (H) qPCR of FOXP1, ALP, COL1A1, HEY1, and HEYL expression in osteogenic cultures of FOXP1-expressing hMPCs in F. n = 3. (I) Model by which FOXP1 regulates BM MSC aging. In young MSCs, high expression of Foxp1 restrains the activation of p16INK4A to maintain the replication capacity of MSCs while sustaining osteogenic rather than adipogenic differentiation. In old MSCs, reduction of Foxp1 expression releases p16INK4A repression, leading to impaired MSC replication capacity while promoting adipogenic differentiation at the expense of osteogenic differentiation. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: For immunofluorescence, heat-induced antigen retrieval with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was performed before bone sections were blocked with 10% normal serum containing 1% BSA in TBST (pH 7.6) for 2 hours at room temperature, then incubated overnight at 4°C with primary antibodies to mouse FOXP1 (Millipore, ABE68, 1:100) and Nestin (Abcam, ab11306, 1:100) diluted in TBST with 1% BSA.

Techniques: In Vitro, Isolation, Transfection, Control, Expressing, Over Expression, Staining, Western Blot, Activation Assay

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 expression in adipocytes is induced by adrenergic stimuli. a H&E staining and immunofluorescence (IF) analysis for the Foxp1 expression in BAT and sWAT from wild type mice at age of 4 weeks. DAPI, blue staining for nucleus; green color for Foxp1 expression. Bar, 50 μm. b Western blotting showed the four isoforms (A, B, D, C) of Foxp1 protein in BAT and sWAT from wild type mice at age of 2 months. c IHC analysis of FOXP1 expression in biopsies from PHEO patients and normal controls. Bar, 10 μm. d qPCR analysis of expression of Foxp1 and brown adipocyte markers ( C/ebpα , Pparγ , and Ucp1 ) during the time course of brown adipocyte differentiation from SVFs. e qPCR analysis of Foxp1 and β3-AR expression in BAT in mice with overnight 4 °C cold exposure. n = 3 biologically independent samples. f Western blotting of Foxp1 in BAT from mice above ( e ). g , h qPCR analysis of Foxp1 and β3-AR expression in brown adipocytes differentiated from murine ( g ) and human SVF ( h ) during an 8-hour CL-316,243 (0.1 μM) treatment as indicated. n = 3 biologically independent experiments. i Foxp1 expression profile in adipocytes derived from 3T3-L1 cells during an 8-h time course, stimulated by CL-316,243 (0.5 μM) with or without SB202190 (p38 kinase inhibitor, 10 μM), FR180204 (Erk1/2 inhibitors, 1 μM) and SCH772984 (Erk1/2 inhibitors, 10 μM), respectively. n = 3 biologically independent experiments. j Western blotting for Foxp1 in brown adipocytes derived from SVF, which were stimulated by CL-316,243 (0.1 μM) with or without SCH772984 (10 μM) for 8 h. * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For western blotting, cells were lysed and protein samples were incubated with primary antibodies against Foxp1 (Millipore, ABE68, 1:1000), C/ebpβ (Santa Cruz, sc-150, 1:500), UCP1(Abcam, ab10893, 1:1000), PGC-1α (Milipore, ab3242 1:1000), p38 (CST, 9212s, 1:1000), phosphorylated p38 (CST, 9211s, 1:1000), HSL (CST, 4107, 1:1000) and phosphorylated HSL (CST, 4126, 1:1000), β3-AR (mybioscience, MBS253490, 1:1000), His-Tag (MBL, M136-3, 1:2000), FLAG (Agilent, 200471, 1:2000) or β-actin (Selleck, A1016,1:2000) at 4 °C overnight.

Techniques: Expressing, Staining, Immunofluorescence, Western Blot, Derivative Assay

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 deficiency promotes brown and beige adipocyte activation. a Representative dorsal view of BAT depot in Foxp1 fl/fl and Foxp1 Myf5 ∆/∆ mice at postnatal day 7. Bar, 1 cm. b HE (upper panel) and IHC staining (lower panel) with UCP1 antibody for BAT in ( a ). Bar, 10 μm. c , d qPCR analysis of Foxp1 and BAT-selective markers for BAT depot. n = 5 biologically independent samples. e Representative view of 3-month-old Foxp1 fl/fl and Foxp1 Ad ∆/∆ mice. Bar, 2 cm. f Representative view of BAT (upper panel) and sWAT depot (lower panel) in ( e ). g HE staining for BAT and sWAT in mutant mice. Bar, 50 μm. h IHC analysis with UCP1 antibody for BAT and sWAT upon 6-h cold exposure. Bar, 50 μm. i – k qPCR for expression of Foxp1 and BAT-selective genes in BAT and sWAT from Foxp1 fl/fl and Foxp1 Ad ∆/∆ mutant mice. n = 4 biologically independent mice/each group. * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For western blotting, cells were lysed and protein samples were incubated with primary antibodies against Foxp1 (Millipore, ABE68, 1:1000), C/ebpβ (Santa Cruz, sc-150, 1:500), UCP1(Abcam, ab10893, 1:1000), PGC-1α (Milipore, ab3242 1:1000), p38 (CST, 9212s, 1:1000), phosphorylated p38 (CST, 9211s, 1:1000), HSL (CST, 4107, 1:1000) and phosphorylated HSL (CST, 4126, 1:1000), β3-AR (mybioscience, MBS253490, 1:1000), His-Tag (MBL, M136-3, 1:2000), FLAG (Agilent, 200471, 1:2000) or β-actin (Selleck, A1016,1:2000) at 4 °C overnight.

Techniques: Activation Assay, Immunohistochemistry, Staining, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 deletion elevates energy expenditure and resists to HFD-induced obesity. a , b VO 2 and VCO 2 of Foxp1 fl/fl and Foxp1 Ad ∆/∆ mice in metabolic cages at age of 3 months old. n = 7 biologically independent mice/each group. c , d Quantification of O 2 and CO 2 consumption in light and dark. e Dorsal view of Foxp1 fl/fl and Foxp1 Ad ∆/∆ mice after 8-week feeding with HFD at age of 6 months old. Bar, 2 cm. f Representative depot of BAT and sWAT in HFD-fed mice ( e ). Bar, 1 cm. g Body weight of HFD-fed mice. h Relative adiposity of HFD-fed mice. i Statistics of rectal temperature of HFD-fed mice. j , k GTT and ITT of HFD-fed mice. * P < 0.05; ** P < 0.01; *** P < 0.001; n = 7 biologically independent mice/each group; error bar, mean ± SEM

Article Snippet: For western blotting, cells were lysed and protein samples were incubated with primary antibodies against Foxp1 (Millipore, ABE68, 1:1000), C/ebpβ (Santa Cruz, sc-150, 1:500), UCP1(Abcam, ab10893, 1:1000), PGC-1α (Milipore, ab3242 1:1000), p38 (CST, 9212s, 1:1000), phosphorylated p38 (CST, 9211s, 1:1000), HSL (CST, 4107, 1:1000) and phosphorylated HSL (CST, 4126, 1:1000), β3-AR (mybioscience, MBS253490, 1:1000), His-Tag (MBL, M136-3, 1:2000), FLAG (Agilent, 200471, 1:2000) or β-actin (Selleck, A1016,1:2000) at 4 °C overnight.

Techniques:

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Overexpression of Foxp1 represses adaptive thermogenesis and is sensitive to HFD-induce obesity. a Representative bioluminescence imaging of two stains of aP2-Foxp1;Ucp1-Luc transgenic mice after 6-hour cold exposure. n = 3 biologically independent mice/each group. Bar, 2 cm. b Representative picture of BAT depot in aP2-Foxp1 transgenic mice. Bar, 0.5 cm. c H&E staining for adipose sections of BAT and sWAT from transgenic mice in ( b ). Bar, 10 μm. d IHC with UCP1 antibody for adipose sections from transgenic mice after 6-h cold exposure. Bar, 10 μm. e Intracellular structure of adipocytes in BAT and sWAT from aP2-Foxp1 mice, as showed by transmission electronic microscope (TEM). Bar, 2 μm. f Assessment of mitochondria DNA abundance by qPCR for the BAT and sWAT from transgenic mice. n = 4 biologically independent mice/each group. g qPCR analysis for Ucp1 expression in adipose tissues of transgenic mice. n = 4 biologically independent samples. h Western blotting for UCP1, HSL, and phosphorylated HSL in BAT and sWAT. i Representative dorsal view of aP2-Foxp1 transgenic mice subjected to 8-week HFD feeding since at age of 3 months. Bar, 2 cm. j Relative adiposity in transgenic mice ( i ). n = 4 biologically independent mice/each group. k The heat map of relative expression of adipogenic genes in three strains of aP2-Foxp1 transgenic mice at age of 3 weeks, as evaluated by RNA-seq. l qPCR analysis validated the BAT-selective genes expression in BAT from ( k ). n = 3 biologically independent mice/each group; * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For western blotting, cells were lysed and protein samples were incubated with primary antibodies against Foxp1 (Millipore, ABE68, 1:1000), C/ebpβ (Santa Cruz, sc-150, 1:500), UCP1(Abcam, ab10893, 1:1000), PGC-1α (Milipore, ab3242 1:1000), p38 (CST, 9212s, 1:1000), phosphorylated p38 (CST, 9211s, 1:1000), HSL (CST, 4107, 1:1000) and phosphorylated HSL (CST, 4126, 1:1000), β3-AR (mybioscience, MBS253490, 1:1000), His-Tag (MBL, M136-3, 1:2000), FLAG (Agilent, 200471, 1:2000) or β-actin (Selleck, A1016,1:2000) at 4 °C overnight.

Techniques: Over Expression, Imaging, Transgenic Assay, Staining, Transmission Assay, Microscopy, Expressing, Western Blot, RNA Sequencing

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 represses β3-AR transcription in adipocytes. a, b β3-AR expression in BAT and sWAT were assessed by qPCR and western blotting in aP2-Foxp1 transgenic mice at 2 month old. n = 3 biologically independent samples/each group. c, d β3-AR expression in BAT and sWAT were assessed by qPCR and western blotting in Foxp1 aP2 ∆/∆ knockout mice at 2 months old. n = 3 biologically independent samples/each group. e ChIP-seq profile showed the Foxp1 binding sites within Adrb3 gene promoter region. Red arrows indicated the potential Foxp1 binding sites. Adrb3 gene was reversely transcribed. ChIP was based on SVF cells derived from BAT. f Upper panel showed the location of Foxp1 binding site at −2251 bp upstream of β3-AR gene promoter with a schematic drawing. ChIP-PCR confirmed the relative enrichment of Foxp1 binding sites (lower panel). n = 3 biologically independent samples/each group. g Basic OCR of SVF-derived brown adipocytes from Foxp1 aP2 ∆/∆ mice, which were transfected β3-AR -shRNA lentivirus or administrated with β3-AR inhibitor L748337 (10 μM) for 1 h. n = 3 biologically independent experiments. h Representative infrared imaging of aP2-Foxp1 transgenic mice with or without 6-hour CL-316,243 (10 μM) exposure. Bar, 1 cm. i Quantification of average skin temperature in boxed regions in ( h ). n = 4 biologically independent mice/each group. Samples were isolated from mice at age of 2 months. j CL-stimulated β3-AR desensitization in SVF-derived brown adipocytes from aP2-Foxp1 transgenic mice and Foxp1 aP2 ∆/∆ knockout mice. β3-AR expression was assessed by qPCR during the 6-h time course of CL-316243 (0.1 μM) treatment. n = 3 biologically independent samples/each group. * P < 0.05; ** P < 0.01; *** P < 0.001; error bar, mean ± SEM

Article Snippet: For western blotting, cells were lysed and protein samples were incubated with primary antibodies against Foxp1 (Millipore, ABE68, 1:1000), C/ebpβ (Santa Cruz, sc-150, 1:500), UCP1(Abcam, ab10893, 1:1000), PGC-1α (Milipore, ab3242 1:1000), p38 (CST, 9212s, 1:1000), phosphorylated p38 (CST, 9211s, 1:1000), HSL (CST, 4107, 1:1000) and phosphorylated HSL (CST, 4126, 1:1000), β3-AR (mybioscience, MBS253490, 1:1000), His-Tag (MBL, M136-3, 1:2000), FLAG (Agilent, 200471, 1:2000) or β-actin (Selleck, A1016,1:2000) at 4 °C overnight.

Techniques: Expressing, Western Blot, Transgenic Assay, Knock-Out, ChIP-sequencing, Binding Assay, Derivative Assay, Transfection, shRNA, Imaging, Isolation

Journal: Nature Communications

Article Title: Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization

doi: 10.1038/s41467-019-12988-8

Figure Lengend Snippet: Foxp1 physically interacts with Prdm16-C/ebpβ complex in brown adipocytes. a , b Co-immunoprecipitation validated the physical interaction between Foxp1, C/ebpβ and Prdm16 proteins in cell lysates from 293T cell lines transfected with indicated plasmids ( a ), or brown adipocytes induced from stromal vascular fractions ( b ). c Immunofluorescence analysis showed the co-localization of Foxp1 and Prdm16 in the nucleus of BAT from wild type mice at age of 1 month old. Bar, 10 μm. d Luciferase reporter assay showed the transactivation of PPARγ -Luc by Foxp1 and Prdm16 protein in 3T3-L1 cell lines. n = 3 biologically independent samples/each group. e Luciferase reporter assay showed the transactivation of β3-AR -Luc by Foxp1, C/ebpβ and Prdm16 protein in 293T cell lines. n = 3 biologically independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001; error bar, mean ± SEM. f Diagram depicting the mechanisms that Foxp1 represses brown/beige adipocyte differentiation and adaptive thermogenesis. Expression of Foxp1 is induced by β3-AR/cAMP/Erk1/2 cascades. Conversely, Foxp1 suppresses β3-AR transcription by counteracting the activity of Prdm16-C/ebpβ complex

Article Snippet: For western blotting, cells were lysed and protein samples were incubated with primary antibodies against Foxp1 (Millipore, ABE68, 1:1000), C/ebpβ (Santa Cruz, sc-150, 1:500), UCP1(Abcam, ab10893, 1:1000), PGC-1α (Milipore, ab3242 1:1000), p38 (CST, 9212s, 1:1000), phosphorylated p38 (CST, 9211s, 1:1000), HSL (CST, 4107, 1:1000) and phosphorylated HSL (CST, 4126, 1:1000), β3-AR (mybioscience, MBS253490, 1:1000), His-Tag (MBL, M136-3, 1:2000), FLAG (Agilent, 200471, 1:2000) or β-actin (Selleck, A1016,1:2000) at 4 °C overnight.

Techniques: Immunoprecipitation, Transfection, Immunofluorescence, Luciferase, Reporter Assay, Expressing, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma

doi: 10.1111/jcmm.18294

Figure Lengend Snippet: The expression of forkhead box protein 1 (FOXP1) in oesophageal cancer and various tumours. (A) The expression of FOXP1 in various tumours in GEPIA database. (B) The expression of FOXP1 in oesophageal cancer tissues and normal tissues in UALCAN database. (C–E) The expression of FOXP1 in oesophageal cancer tissues depending on pathological type, tumour grade and tumour stage. (F–I) The expression of FOXP1 in oesophageal squamous cell carcinoma in GSE17351, GSE70409, GSE77861 and GSE100942 datasets. (J–K) The expression of FOXP1 in oesophageal squamous cell carcinoma tissues and adjacent normal tissues. * p < 0.05.

Article Snippet: FOXP1 primary antibody (1:150 dilution, BOSTER, M00723‐4) was added and incubated at room temperature for 1 h. The slices were incubated with the secondary antibody for 30 min at room temperature after washing with PBS buffer.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma

doi: 10.1111/jcmm.18294

Figure Lengend Snippet: High expression of forkhead box protein 1 (FOXP1) indicates a better survival prognosis in oesophageal cancer. (A) The correlation between expression of FOXP1 and overall survival prognosis in different tumours in TISCH2 database. (B) Kaplan–Meier survival curves of overall survival for oesophageal cancer patients with high and low expression of FOXP1 in GEPIA database. (C) Kaplan–Meier survival curves of disease free survival for oesophageal cancer patients with high and low expression of FOXP1 in GEPIA database. (D) Kaplan–Meier survival curves of overall survival for oesophageal cancer patients with high and low expression of FOXP1 in KM plotter database. (E) Kaplan–Meier survival curves of disease free survival for oesophageal cancer patients with high and low expression of FOXP1 in KM plotter database. (F) Kaplan–Meier survival curves of overall survival for oesophageal squamous cell carcinoma patients with high and low expression of FOXP1 in KM plotter database. (G) Kaplan–Meier survival curves of overall survival for oesophageal squamous cell carcinoma patients with high and low expression of FOXP1 in GSE53625 dataset.

Article Snippet: FOXP1 primary antibody (1:150 dilution, BOSTER, M00723‐4) was added and incubated at room temperature for 1 h. The slices were incubated with the secondary antibody for 30 min at room temperature after washing with PBS buffer.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma

doi: 10.1111/jcmm.18294

Figure Lengend Snippet: Identification of differential expression genes in high and low expression of forkhead box protein 1 (FOXP1) cohorts in oesophageal squamous cell carcinoma. (A) Venn diagram showing the intersection of differential expression genes in GSE26886 and GSE45168 datasets. (B, C) Volcano plots showing the differential expression genes in GSE26886 and GSE45168 datasets. (D, E) Cluster heatmaps of genes in GSE26886 and GSE45168 datasets.

Article Snippet: FOXP1 primary antibody (1:150 dilution, BOSTER, M00723‐4) was added and incubated at room temperature for 1 h. The slices were incubated with the secondary antibody for 30 min at room temperature after washing with PBS buffer.

Techniques: Quantitative Proteomics, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma

doi: 10.1111/jcmm.18294

Figure Lengend Snippet: Forkhead box protein 1 (FOXP1) functional analysis and interaction network construction. (A) Gene ontology enrichment analysis for differential expression genes. (B) KEGG enrichment analysis for differential expression genes. (C) Network of enriched terms in metascape database. (D) The cord diagram of the correspondence between differential expression genes and signalling pathways. (E, F) Protein–protein interaction (PPI) network and MCODE components identified in differential expression genes in Metascape database. (G) The gene co‐expression network for FOXP1 in GeneMANIA database. (H) PPI network of differential expression genes in STRING database.

Article Snippet: FOXP1 primary antibody (1:150 dilution, BOSTER, M00723‐4) was added and incubated at room temperature for 1 h. The slices were incubated with the secondary antibody for 30 min at room temperature after washing with PBS buffer.

Techniques: Functional Assay, Quantitative Proteomics, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma

doi: 10.1111/jcmm.18294

Figure Lengend Snippet: Association of forkhead box protein 1 (FOXP1) with the tumour immune microenvironment in oesophageal cancer. (A) The association of FOXP1 expression and tumour immune infiltration cells level in oesophageal cancer in TIMER database. (B) The relative proportion of different somatic copy number alterations (SCNA) states of FOXP1 for various TCGA tumour types in TIMER2.0 database. (C) The relationship between different SCNA states of FOXP1 and infiltration level of six types of immune cells in oesophageal cancer. * p < 0.05. (D) The relative proportion of the infiltration levels of 22 immune infiltrating cells in oesophageal squamous cell carcinoma based on GSE75241 dataset. (E) The immune cells correlation heatmap in oesophageal squamous cell carcinoma based on GSE75241 dataset.

Article Snippet: FOXP1 primary antibody (1:150 dilution, BOSTER, M00723‐4) was added and incubated at room temperature for 1 h. The slices were incubated with the secondary antibody for 30 min at room temperature after washing with PBS buffer.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: An integrated analysis of the anticarcinogenic role of forkhead box protein 1 in oesophageal squamous cell carcinoma

doi: 10.1111/jcmm.18294

Figure Lengend Snippet: Forkhead box protein 1 (FOXP1) inhibits proliferation of oesophageal squamous cell carcinoma cells. (A, B) Western blot assay confirmed the expression levels of FOXP1 protein in different oesophageal squamous cell carcinoma cell lines and oesophageal epithelial cells. * p < 0.05, ** p < 0.01, ns p > 0.05. (C, D) Western blot assay confirmed the transfection efficiency of overexpression of FOXP1 in EC109 cells. ** p < 0.01. (E) The CCK‐8 assay showed overexpression of FOXP1 inhibits the proliferation of EC109 cells. *** p < 0.001. (F, G) The colony formation assay showed overexpression of FOXP1 inhibits the colony forming ability of EC109 cells. (H, I) The subcutaneous tumour formation assay showed overexpression of FOXP1 inhibits the ability of tumorigenicity of EC109 cells in vivo. * p < 0.05, ** p < 0.01, ns p > 0.05.

Article Snippet: FOXP1 primary antibody (1:150 dilution, BOSTER, M00723‐4) was added and incubated at room temperature for 1 h. The slices were incubated with the secondary antibody for 30 min at room temperature after washing with PBS buffer.

Techniques: Western Blot, Expressing, Transfection, Over Expression, CCK-8 Assay, Colony Assay, Tube Formation Assay, In Vivo

Journal: Cell Cycle

Article Title: Down-regulated microRNA-183 mediates the Jak/Stat signaling pathway to attenuate hippocampal neuron injury in epilepsy rats by targeting Foxp1

doi: 10.1080/15384101.2019.1671717

Figure Lengend Snippet: Primer sequence.

Article Snippet: The proteins were added with primary antibodies: Foxp1 (1:100), Jak1 (1:500), Stat1 (1:500) and Stat3 (1:500, all from Abcam, Cambridge, UK), Bcl-2 and Bax (1:500, both from Proteintech, Chicago, USA), incubated overnight.

Techniques: Sequencing

Journal: Aging (Albany NY)

Article Title: CASC21, a FOXP1 induced long non-coding RNA, promotes colorectal cancer growth by regulating CDK6

doi: 10.18632/aging.103376

Figure Lengend Snippet: FOXP1 induces CASC21 high expression in CRC. ( A ) FOXP1 expression was detected by qRT-PCR in 80 pairs of CRC and corresponding adjacent normal tissues. ( B ) CASC21 expression was detected in HCT-116 and HCT-8 cells transfected with FOXP1 siRNAs or FOXP1 overexpression vector by qRT-PCR. ( C ) The correlation between FOXP1 and CASC21 expression analyzed in 80 paired CRC samples (n= 80, r= 0.483, P< 0.001). ( D ) ChIP assays were conducted to identify FOXP1 occupancy in the CASC21 promoter region. ( E ) Luciferase reporter assays were used to determine the FOXP1 binding sites on the CASC21 promoter region. All data represent mean ± SEM (n = 3-6). ** P < 0.01 and *** P < 0.001.

Article Snippet: The membranes were blocked with 5% skim milk for 1 h and then incubated with primary antibodies (anti-FOXP1 (#4402, Cell Signaling Technology, USA), anti-Cyclin D1(#55506, Cell Signaling Technology, USA), anti-Cyclin D2(#3741, Cell Signaling Technology, USA) anti- CDK6 (#13331, Cell Signaling Technology, USA) and anti-GAPDH(#5174, Cell Signaling Technology, USA)) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Over Expression, Plasmid Preparation, Luciferase, Binding Assay

Journal: Diagnostic Pathology

Article Title: Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

doi: 10.1186/s13000-018-0715-y

Figure Lengend Snippet: FOXP1 protein expression patterns in different breast lesions

Article Snippet: Following deparaffinization and heat-mediated antigen retrieval, immunohistochemical staining was carried out using an Envision system (DAKO, Glostrup, Denmark) with primary antibodies against FOXP1 (JC12, AbD Serotec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), calpain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, Roche Tucson), pAKT (736E11, Cell signaling, Danvers, MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 (53H11, Cell signaling) and p-p70S6K (49D7, Cell signaling).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

doi: 10.1186/s13000-018-0715-y

Figure Lengend Snippet: FOXP1 expression patterns in DCIS. FOXP1 immunostaining was observed in the nuclei of tumor cells ( a ×200, b ×400) or both the nuclei and cytoplasm ( c ×200, d ×400)

Article Snippet: Following deparaffinization and heat-mediated antigen retrieval, immunohistochemical staining was carried out using an Envision system (DAKO, Glostrup, Denmark) with primary antibodies against FOXP1 (JC12, AbD Serotec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), calpain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, Roche Tucson), pAKT (736E11, Cell signaling, Danvers, MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 (53H11, Cell signaling) and p-p70S6K (49D7, Cell signaling).

Techniques: Expressing, Immunostaining

Journal: Diagnostic Pathology

Article Title: Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

doi: 10.1186/s13000-018-0715-y

Figure Lengend Snippet: FOXP1 expression patterns in IDC. The FOXP1 protein expression patterns in IDC tumor cells ranged from exclusive cytoplasmic ( a , TMA; b ×400) to mixed nuclear/cytoplasmic ( c , TMA; d ×400) and to exclusive nuclear ( e , TMA; f ×400)

Article Snippet: Following deparaffinization and heat-mediated antigen retrieval, immunohistochemical staining was carried out using an Envision system (DAKO, Glostrup, Denmark) with primary antibodies against FOXP1 (JC12, AbD Serotec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), calpain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, Roche Tucson), pAKT (736E11, Cell signaling, Danvers, MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 (53H11, Cell signaling) and p-p70S6K (49D7, Cell signaling).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

doi: 10.1186/s13000-018-0715-y

Figure Lengend Snippet: The correlation between cytoplasmic FOXP1 expression and clinicopathological parameters in IDC cases

Article Snippet: Following deparaffinization and heat-mediated antigen retrieval, immunohistochemical staining was carried out using an Envision system (DAKO, Glostrup, Denmark) with primary antibodies against FOXP1 (JC12, AbD Serotec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), calpain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, Roche Tucson), pAKT (736E11, Cell signaling, Danvers, MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 (53H11, Cell signaling) and p-p70S6K (49D7, Cell signaling).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Cytoplasmic FOXP1 expression is correlated with ER and calpain II expression and predicts a poor outcome in breast cancer

doi: 10.1186/s13000-018-0715-y

Figure Lengend Snippet: Kaplan-Meier survival curves of patients with IDC according to C-FOXP1 expression. Patients with positive C-FOXP1 immunoreactivity showed inferior OS ( a ) and DFS ( b ) compared with C-FOXP1-negative patients, although the difference in DFS was not statistically significant

Article Snippet: Following deparaffinization and heat-mediated antigen retrieval, immunohistochemical staining was carried out using an Envision system (DAKO, Glostrup, Denmark) with primary antibodies against FOXP1 (JC12, AbD Serotec, Oxford, UK), ER (SP1, Roche Tucson, AZ, USA), calpain II (CAPN2, Sigma, St. Louis, MO, USA), HER2 (4B5, Roche Tucson), pAKT (736E11, Cell signaling, Danvers, MA, USA), pmTOR (49F9, Cell signaling), p4E-BP1 (53H11, Cell signaling) and p-p70S6K (49D7, Cell signaling).

Techniques: Expressing