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10 ng/ml mouse egf  (PeproTech)

 
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    PeproTech 10 ng/ml mouse egf
    (A) qRT-PCR analysis of FOXG1 transgene and endogenous FoxO6 expression in two independent adult mouse NSC lines (‘F6’ and ‘F11-19’) with Dox-inducible FOXG1-V5 expression grown in NSC media with or without Dox for 24 h. Expression shown relative to -Dox (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments, respectively. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05. (B) Schematic of HDR-mediated knock-in of an HA epitope tag at the 3’ end of last FoxO6 coding exon in F6 cells. PCR genotyping of the bulk transfected F6 cell population revealed a 196 bp product, indicating the presence of cells with insertion of the HA tag at the 3’ end of FoxO6. (C) Wide-field immunofluorescent images following immunocytochemistry (ICC) of FoxO6-HA (red) and DAPI (blue) in the tagged F6 NSCs following Dox addition for 4 days <t>in</t> <t>EGF/FGF-2.</t> Scale bar: 100 μm. (D) Western immunoblot analysis of FOXG1-V5 and FoxO6-HA protein expression in tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. GAPDH was used as a loading control. (E) Experimental strategy for Foxg1 deletion in IENS-GFP cells. Yellow triangles show the target sites of the sgRNAs at either the 5’ or 3’ end of the coding exon. PCR genotyping of parental IENS-GFP cells and Foxg1 KO clonal cell lines (KO 58 and KO 59). Wildtype PCR product ~2.6 kb, knockout PCR product ~1.3 kb. (F) ICC analysis confirms loss of FOXG1 protein expression in IENS-GFP Foxg1 KO clonal lines (KO 58 and KO 59). Scale bar: 100 μm. (G) Western immunoblot confirming loss of Foxg1 protein in the two independent IENS-GFP knock-out clonal lines (KO 58 and KO 59). GAPDH was used as a loading control. (H) qRT-PCR analysis of FoxO6 expression in IENS-GFP Foxg1 knock-out clonal lines, compared to parental IENS-GFP (in which log 2 (FC) = 0). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05.
    10 Ng/Ml Mouse Egf, supplied by PeproTech, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/10 ng/ml mouse egf/product/PeproTech
    Average 90 stars, based on 1 article reviews
    10 ng/ml mouse egf - by Bioz Stars, 2026-03
    90/100 stars

    Images

    1) Product Images from "Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6"

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    Journal: bioRxiv

    doi: 10.1101/2022.06.30.498283

    (A) qRT-PCR analysis of FOXG1 transgene and endogenous FoxO6 expression in two independent adult mouse NSC lines (‘F6’ and ‘F11-19’) with Dox-inducible FOXG1-V5 expression grown in NSC media with or without Dox for 24 h. Expression shown relative to -Dox (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments, respectively. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05. (B) Schematic of HDR-mediated knock-in of an HA epitope tag at the 3’ end of last FoxO6 coding exon in F6 cells. PCR genotyping of the bulk transfected F6 cell population revealed a 196 bp product, indicating the presence of cells with insertion of the HA tag at the 3’ end of FoxO6. (C) Wide-field immunofluorescent images following immunocytochemistry (ICC) of FoxO6-HA (red) and DAPI (blue) in the tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. Scale bar: 100 μm. (D) Western immunoblot analysis of FOXG1-V5 and FoxO6-HA protein expression in tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. GAPDH was used as a loading control. (E) Experimental strategy for Foxg1 deletion in IENS-GFP cells. Yellow triangles show the target sites of the sgRNAs at either the 5’ or 3’ end of the coding exon. PCR genotyping of parental IENS-GFP cells and Foxg1 KO clonal cell lines (KO 58 and KO 59). Wildtype PCR product ~2.6 kb, knockout PCR product ~1.3 kb. (F) ICC analysis confirms loss of FOXG1 protein expression in IENS-GFP Foxg1 KO clonal lines (KO 58 and KO 59). Scale bar: 100 μm. (G) Western immunoblot confirming loss of Foxg1 protein in the two independent IENS-GFP knock-out clonal lines (KO 58 and KO 59). GAPDH was used as a loading control. (H) qRT-PCR analysis of FoxO6 expression in IENS-GFP Foxg1 knock-out clonal lines, compared to parental IENS-GFP (in which log 2 (FC) = 0). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05.
    Figure Legend Snippet: (A) qRT-PCR analysis of FOXG1 transgene and endogenous FoxO6 expression in two independent adult mouse NSC lines (‘F6’ and ‘F11-19’) with Dox-inducible FOXG1-V5 expression grown in NSC media with or without Dox for 24 h. Expression shown relative to -Dox (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments, respectively. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05. (B) Schematic of HDR-mediated knock-in of an HA epitope tag at the 3’ end of last FoxO6 coding exon in F6 cells. PCR genotyping of the bulk transfected F6 cell population revealed a 196 bp product, indicating the presence of cells with insertion of the HA tag at the 3’ end of FoxO6. (C) Wide-field immunofluorescent images following immunocytochemistry (ICC) of FoxO6-HA (red) and DAPI (blue) in the tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. Scale bar: 100 μm. (D) Western immunoblot analysis of FOXG1-V5 and FoxO6-HA protein expression in tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. GAPDH was used as a loading control. (E) Experimental strategy for Foxg1 deletion in IENS-GFP cells. Yellow triangles show the target sites of the sgRNAs at either the 5’ or 3’ end of the coding exon. PCR genotyping of parental IENS-GFP cells and Foxg1 KO clonal cell lines (KO 58 and KO 59). Wildtype PCR product ~2.6 kb, knockout PCR product ~1.3 kb. (F) ICC analysis confirms loss of FOXG1 protein expression in IENS-GFP Foxg1 KO clonal lines (KO 58 and KO 59). Scale bar: 100 μm. (G) Western immunoblot confirming loss of Foxg1 protein in the two independent IENS-GFP knock-out clonal lines (KO 58 and KO 59). GAPDH was used as a loading control. (H) qRT-PCR analysis of FoxO6 expression in IENS-GFP Foxg1 knock-out clonal lines, compared to parental IENS-GFP (in which log 2 (FC) = 0). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05.

    Techniques Used: Quantitative RT-PCR, Expressing, Two Tailed Test, Knock-In, Transfection, Immunocytochemistry, Western Blot, Knock-Out

    (A) Schematic of the experimental design for assessing FOXG1-induced reactivation of quiescent NSCs and associated changes in gene expression, using clonal ‘F6’ adult mouse NSC line with Dox-inducible FOXG1-V5 expression. Non-BMP4 treated control = cells in NSC media with EGF/FGF-2. (B) ICC for V5, confirming FOXG1-V5 expression upon Dox addition (Scale bar: 100 μm). (C) Representative phase-contrast images showing changes in cell morphology upon addition of Dox (Scale bar: 100 μm). (D) Colony formation after 24 h BMP4 treatment followed by 10 days in EGF/FGF-2 with or without Dox. Representative images shown of wells stained with methylene blue and imaged on a bright-field microscope. n=3 independent experiments. (E) Higher magnification phase-contrast images of representative colonies after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox as in panel (D) (Scale bar: 200 μm). (F) Number of colonies formed after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox. Mean +/− SD, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (G) qRT-PCR analysis of human FOXG1 transgene and (H) FoxO6 expression during the reactivation time course. Pink = + Dox addition, Blue = No Dox addition. Expression shown relative to non BMP4-treated (EGF/FGF-2) control (in which log 2 (FC)= 0 (dotted line)). Day 0 = expression after 24 h BMP4 treatment. Mean +/− SEM. n = 2 ( FOXG1 ) or 4 ( FoxO6 ). Each data point shows the mean of one experiment performed in technical duplicates. **** P≤0.0001. Two way Anova with Sidak correction.
    Figure Legend Snippet: (A) Schematic of the experimental design for assessing FOXG1-induced reactivation of quiescent NSCs and associated changes in gene expression, using clonal ‘F6’ adult mouse NSC line with Dox-inducible FOXG1-V5 expression. Non-BMP4 treated control = cells in NSC media with EGF/FGF-2. (B) ICC for V5, confirming FOXG1-V5 expression upon Dox addition (Scale bar: 100 μm). (C) Representative phase-contrast images showing changes in cell morphology upon addition of Dox (Scale bar: 100 μm). (D) Colony formation after 24 h BMP4 treatment followed by 10 days in EGF/FGF-2 with or without Dox. Representative images shown of wells stained with methylene blue and imaged on a bright-field microscope. n=3 independent experiments. (E) Higher magnification phase-contrast images of representative colonies after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox as in panel (D) (Scale bar: 200 μm). (F) Number of colonies formed after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox. Mean +/− SD, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (G) qRT-PCR analysis of human FOXG1 transgene and (H) FoxO6 expression during the reactivation time course. Pink = + Dox addition, Blue = No Dox addition. Expression shown relative to non BMP4-treated (EGF/FGF-2) control (in which log 2 (FC)= 0 (dotted line)). Day 0 = expression after 24 h BMP4 treatment. Mean +/− SEM. n = 2 ( FOXG1 ) or 4 ( FoxO6 ). Each data point shows the mean of one experiment performed in technical duplicates. **** P≤0.0001. Two way Anova with Sidak correction.

    Techniques Used: Expressing, Staining, Microscopy, Quantitative RT-PCR

    (A) Schematic of FoxO6 locus following CRISPR/Cas9-mediated knockout strategy. Yellow triangles show the sgRNA target sites, resulting in a 178 bp deletion in allele 1 in FoxO6 KO clone 53. Exon 1 of allele 2 is replaced by an EF1a-puromycin cassette. (B) PCR genotyping of FoxO6 KO clonal cell lines 6, 53 and 62. PCR 1 and PCR 2, across the 5’ and 3’ homology arms of the EF1a-puromycin cassette, respectively, show correct integration at one of the FoxO6 alleles. PCR 3 shows a 178 bp deletion (53) or loss (6, 62) of the remaining FoxO6 allele. WT parental band = 565 bp, knockout (53) band = 387 bp. (C) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell lines 6 and 62, compared to parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (D) EdU incorporation assay (24h pulse) in parental and FoxO6 KO clonal lines (6 and 62) grown in EGF/FGF-2. (Left) Representative fluorescent images of EdU incorporation. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle marker ( Cdk4, Plk1, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) marker expression in ANS4 parental and FoxO6 KO clonal cell lines (6 and 62) in EGF/FGF-2 and after 24 h BMP4 treatment. Expression values were normalised to Gapdh and shown relative to the expression in parental cells in EGF/FGF-2 (in which log 2 (FC) = 0, shown by the dotted line). Graph shows Mean +/− SD. One experiment, performed in technical duplicates.
    Figure Legend Snippet: (A) Schematic of FoxO6 locus following CRISPR/Cas9-mediated knockout strategy. Yellow triangles show the sgRNA target sites, resulting in a 178 bp deletion in allele 1 in FoxO6 KO clone 53. Exon 1 of allele 2 is replaced by an EF1a-puromycin cassette. (B) PCR genotyping of FoxO6 KO clonal cell lines 6, 53 and 62. PCR 1 and PCR 2, across the 5’ and 3’ homology arms of the EF1a-puromycin cassette, respectively, show correct integration at one of the FoxO6 alleles. PCR 3 shows a 178 bp deletion (53) or loss (6, 62) of the remaining FoxO6 allele. WT parental band = 565 bp, knockout (53) band = 387 bp. (C) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell lines 6 and 62, compared to parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (D) EdU incorporation assay (24h pulse) in parental and FoxO6 KO clonal lines (6 and 62) grown in EGF/FGF-2. (Left) Representative fluorescent images of EdU incorporation. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle marker ( Cdk4, Plk1, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) marker expression in ANS4 parental and FoxO6 KO clonal cell lines (6 and 62) in EGF/FGF-2 and after 24 h BMP4 treatment. Expression values were normalised to Gapdh and shown relative to the expression in parental cells in EGF/FGF-2 (in which log 2 (FC) = 0, shown by the dotted line). Graph shows Mean +/− SD. One experiment, performed in technical duplicates.

    Techniques Used: CRISPR, Knock-Out, Quantitative RT-PCR, Expressing, Marker

    (A) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell line 53, compared to ANS4 parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (B) Representative phase-contrast images showing typical NSC morphology in parental and FoxO6 KO 53 cells. Scale bar: 25 μm. (C) Growth curve analysis of parental and FoxO6 KO 53 clonal cells in EGF/FGF-2. Light grey = parental, black = FoxO6 KO 53. Mean +/− SD, n=3 technical replicates. Representative of n=4 independent experiments. Graph showing the gradient of the linear portion of the logistic growth curve (% / h). Mean +/− SEM, n=4 independent experiments. Two-tailed paired Student’s t-test. * P ≤ 0.05. (D) EdU incorporation assay (24h pulse) in EGF/FGF-2 for parental and FoxO6 KO 53 cells. (Left) Representative fluorescent images of EdU incorporation after 24 h pulse. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) Brightfield images of colony formation by parental or FoxO6 KO 53 cells 10 days after plating at low density in NSC media (EGF/FGF-2). Plates stained with methylene blue. (F) Representative phase-contrast images showing morphology of ANS4 and FoxO6 KO 53 after 24h BMP4 treatment. Scale bar: 25 μm. (G) ICC analysis of Nestin and Gfap expression in ANS4 parental and FoxO6 KO 53 cells in EGF/FGF-2 or 3 days BMP4 treatment at low density. Scale bar: 100 μm. (H) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle ( Plk1, Cdk4, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) markers, in parental and FoxO6 KO 53 cells in EGF/FGF-2 and after 24 h BMP4 treatment. Expression shown relative to parental in EGF/FGF-2 (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. (I) EdU incorporation after treatment with BMP4 or EGF/FGF-2 for 24h, followed by a 24h EdU pulse in EGF/FGF-2. Mean +/− SEM. n=2 independent experiments. Each data point shows the mean one experiment, performed in technical triplicates.
    Figure Legend Snippet: (A) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell line 53, compared to ANS4 parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (B) Representative phase-contrast images showing typical NSC morphology in parental and FoxO6 KO 53 cells. Scale bar: 25 μm. (C) Growth curve analysis of parental and FoxO6 KO 53 clonal cells in EGF/FGF-2. Light grey = parental, black = FoxO6 KO 53. Mean +/− SD, n=3 technical replicates. Representative of n=4 independent experiments. Graph showing the gradient of the linear portion of the logistic growth curve (% / h). Mean +/− SEM, n=4 independent experiments. Two-tailed paired Student’s t-test. * P ≤ 0.05. (D) EdU incorporation assay (24h pulse) in EGF/FGF-2 for parental and FoxO6 KO 53 cells. (Left) Representative fluorescent images of EdU incorporation after 24 h pulse. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) Brightfield images of colony formation by parental or FoxO6 KO 53 cells 10 days after plating at low density in NSC media (EGF/FGF-2). Plates stained with methylene blue. (F) Representative phase-contrast images showing morphology of ANS4 and FoxO6 KO 53 after 24h BMP4 treatment. Scale bar: 25 μm. (G) ICC analysis of Nestin and Gfap expression in ANS4 parental and FoxO6 KO 53 cells in EGF/FGF-2 or 3 days BMP4 treatment at low density. Scale bar: 100 μm. (H) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle ( Plk1, Cdk4, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) markers, in parental and FoxO6 KO 53 cells in EGF/FGF-2 and after 24 h BMP4 treatment. Expression shown relative to parental in EGF/FGF-2 (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. (I) EdU incorporation after treatment with BMP4 or EGF/FGF-2 for 24h, followed by a 24h EdU pulse in EGF/FGF-2. Mean +/− SEM. n=2 independent experiments. Each data point shows the mean one experiment, performed in technical triplicates.

    Techniques Used: Quantitative RT-PCR, Expressing, Two Tailed Test, Staining

    (A) qRT-PCR analysis of FOXG1 transgene expression in parental or FoxO6 KO 53 cells engineered with inducible FOXG1-V5 after 24 h BMP4 and return to EGF/FGF-2 with our without Dox for 4 days. Expression shown relative parental non-BMP treated (EGF/FGF-2) control (in which log 2 (FC) = 0 (dotted line)). Mean +/− SEM. n=5 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed paired t-test. (B) Representative ICC images showing FOXG1-V5 expression after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox, in parental and FoxO6 KO 53 cells with inducible FOXG1-V5. Scale bar: 100 μm. (C) Percentage of parental or FoxO6 KO 53 cells with inducible FOXG1 expressing FOXG1-V5 (assessed by ICC) after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox. Mean +/− SEM. n = 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Twotailed paired t-test. (D) Representative images of colony formation assay with parental and FoxO6 KO 53 cells at day 10 in EGF/FGF-2, with or without Dox. Plates stained with methylene blue following fixation. (E) Numbers of colonies formed after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM, n= 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Two tailed paired Student’s t-tests. ** P ≤ 0.01. (F) Percentage of the well area covered by cells after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM. n=4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates.
    Figure Legend Snippet: (A) qRT-PCR analysis of FOXG1 transgene expression in parental or FoxO6 KO 53 cells engineered with inducible FOXG1-V5 after 24 h BMP4 and return to EGF/FGF-2 with our without Dox for 4 days. Expression shown relative parental non-BMP treated (EGF/FGF-2) control (in which log 2 (FC) = 0 (dotted line)). Mean +/− SEM. n=5 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed paired t-test. (B) Representative ICC images showing FOXG1-V5 expression after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox, in parental and FoxO6 KO 53 cells with inducible FOXG1-V5. Scale bar: 100 μm. (C) Percentage of parental or FoxO6 KO 53 cells with inducible FOXG1 expressing FOXG1-V5 (assessed by ICC) after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox. Mean +/− SEM. n = 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Twotailed paired t-test. (D) Representative images of colony formation assay with parental and FoxO6 KO 53 cells at day 10 in EGF/FGF-2, with or without Dox. Plates stained with methylene blue following fixation. (E) Numbers of colonies formed after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM, n= 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Two tailed paired Student’s t-tests. ** P ≤ 0.01. (F) Percentage of the well area covered by cells after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM. n=4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates.

    Techniques Used: Quantitative RT-PCR, Expressing, Two Tailed Test, Colony Assay, Staining

    (A) Phase contrast imaging following Dox addition to untransfected ANS4 cells does not induce vacuole formation. Scale bar 100 um. (B) ICC following Dox-induced (24h) MYEF2-HA-IRES-MCHERRY overexpression in mouse GSC line ‘NPE’ shows no evidence of vacuole formation. Scale bar 50 um. (C) BODIPY lipid staining does not colocalise with vacuole structures in FoxO6-inducible cell line (C71, 2 days +/− Dox). Scale bar 25um. (D) EGF-647 uptake after a pulse of 1 hr shows puncta representative of receptor-mediated endocytosis (C71 incubated overnight with Dox prior to EGF-647 pulse). Scale bar 25 um. (E) Western blot analysis of LAMP1, EEA1 and HA upon FoxO6-HA overexpression (+/− Dox). GAPDH is used as a loading control. Bulk transfected population sorted for mCherry and clonal cell lines (6, 41, 59, 71) analysed. (F) Phase contrast images show vacuole formation in FoxO6-HA inducible cell lines following Dox+Dextran overnight incubation, prior to flow cytometry analysis. Scale bar 100 um. (G) Imaging of EdU incorporation in FoxO6-HA inducible cells (C71) after 2 days in EGF/FGF +/− Dox (24h pulse). Scale bar 100 um or 25um. (H) EdU incorporation after EGF/FGF-2 or BMP4 for 3 days +/− Dox (24h pulse). n=3 technical replicates, mean +/− SD. (I) Phase-contrast images of Dox treated cells in culture (c71). Vacuolated cells remain after 5 days in Dox and following Dox removal. (J) ICC in F6 cells with Dox-inducible FOXG1-V5 shows no evidence of vacuolisation upon Dox addition (FOXG1-V5 induction). Scale bar 100 um.
    Figure Legend Snippet: (A) Phase contrast imaging following Dox addition to untransfected ANS4 cells does not induce vacuole formation. Scale bar 100 um. (B) ICC following Dox-induced (24h) MYEF2-HA-IRES-MCHERRY overexpression in mouse GSC line ‘NPE’ shows no evidence of vacuole formation. Scale bar 50 um. (C) BODIPY lipid staining does not colocalise with vacuole structures in FoxO6-inducible cell line (C71, 2 days +/− Dox). Scale bar 25um. (D) EGF-647 uptake after a pulse of 1 hr shows puncta representative of receptor-mediated endocytosis (C71 incubated overnight with Dox prior to EGF-647 pulse). Scale bar 25 um. (E) Western blot analysis of LAMP1, EEA1 and HA upon FoxO6-HA overexpression (+/− Dox). GAPDH is used as a loading control. Bulk transfected population sorted for mCherry and clonal cell lines (6, 41, 59, 71) analysed. (F) Phase contrast images show vacuole formation in FoxO6-HA inducible cell lines following Dox+Dextran overnight incubation, prior to flow cytometry analysis. Scale bar 100 um. (G) Imaging of EdU incorporation in FoxO6-HA inducible cells (C71) after 2 days in EGF/FGF +/− Dox (24h pulse). Scale bar 100 um or 25um. (H) EdU incorporation after EGF/FGF-2 or BMP4 for 3 days +/− Dox (24h pulse). n=3 technical replicates, mean +/− SD. (I) Phase-contrast images of Dox treated cells in culture (c71). Vacuolated cells remain after 5 days in Dox and following Dox removal. (J) ICC in F6 cells with Dox-inducible FOXG1-V5 shows no evidence of vacuolisation upon Dox addition (FOXG1-V5 induction). Scale bar 100 um.

    Techniques Used: Imaging, Over Expression, Staining, Incubation, Western Blot, Transfection, Flow Cytometry

    (A) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells with Dox-inducible FOXG1-V5 grown in EGF/FGF-2 for 24h plus or minus Dox (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates). (B) Western blot analysis of Pak1 expression in F6 cells treated with Dox in EGF/FGF for 24h. GAPDH is used as a loading control. Quantification of Pak1 bands normalised to GAPDH and -Dox control shown, where -Dox =1. (C) qRT-PCR analysis of Pak1 expression in ANS4 parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates.) Two-tailed one sample t-test, * p<0.05. (D) Western blot analysis of Pak1 expression in parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates). (E) Quantification of Pak1 Western blot band intensities as in panel (D). Parental = 1 as shown by the dotted line (n=3 biological replicates, Mean +/− SEM. Each data point shows the intensity from one experiment). Two-tailed one sample t-test, * p<0.05. (F) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells after 24 h BMP4 and return to EGF/FGF-2 with or without Dox for 2 days. Expression shown relative non BMP-treated (EGF/FGF-2) control (in which log 2 (FC) = 0). Day 0 = expression after 24 h BMP4 treatment (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates).
    Figure Legend Snippet: (A) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells with Dox-inducible FOXG1-V5 grown in EGF/FGF-2 for 24h plus or minus Dox (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates). (B) Western blot analysis of Pak1 expression in F6 cells treated with Dox in EGF/FGF for 24h. GAPDH is used as a loading control. Quantification of Pak1 bands normalised to GAPDH and -Dox control shown, where -Dox =1. (C) qRT-PCR analysis of Pak1 expression in ANS4 parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates.) Two-tailed one sample t-test, * p<0.05. (D) Western blot analysis of Pak1 expression in parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates). (E) Quantification of Pak1 Western blot band intensities as in panel (D). Parental = 1 as shown by the dotted line (n=3 biological replicates, Mean +/− SEM. Each data point shows the intensity from one experiment). Two-tailed one sample t-test, * p<0.05. (F) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells after 24 h BMP4 and return to EGF/FGF-2 with or without Dox for 2 days. Expression shown relative non BMP-treated (EGF/FGF-2) control (in which log 2 (FC) = 0). Day 0 = expression after 24 h BMP4 treatment (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates).

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot, Clone Assay, Two Tailed Test



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    Image Search Results


    (A) qRT-PCR analysis of FOXG1 transgene and endogenous FoxO6 expression in two independent adult mouse NSC lines (‘F6’ and ‘F11-19’) with Dox-inducible FOXG1-V5 expression grown in NSC media with or without Dox for 24 h. Expression shown relative to -Dox (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments, respectively. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05. (B) Schematic of HDR-mediated knock-in of an HA epitope tag at the 3’ end of last FoxO6 coding exon in F6 cells. PCR genotyping of the bulk transfected F6 cell population revealed a 196 bp product, indicating the presence of cells with insertion of the HA tag at the 3’ end of FoxO6. (C) Wide-field immunofluorescent images following immunocytochemistry (ICC) of FoxO6-HA (red) and DAPI (blue) in the tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. Scale bar: 100 μm. (D) Western immunoblot analysis of FOXG1-V5 and FoxO6-HA protein expression in tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. GAPDH was used as a loading control. (E) Experimental strategy for Foxg1 deletion in IENS-GFP cells. Yellow triangles show the target sites of the sgRNAs at either the 5’ or 3’ end of the coding exon. PCR genotyping of parental IENS-GFP cells and Foxg1 KO clonal cell lines (KO 58 and KO 59). Wildtype PCR product ~2.6 kb, knockout PCR product ~1.3 kb. (F) ICC analysis confirms loss of FOXG1 protein expression in IENS-GFP Foxg1 KO clonal lines (KO 58 and KO 59). Scale bar: 100 μm. (G) Western immunoblot confirming loss of Foxg1 protein in the two independent IENS-GFP knock-out clonal lines (KO 58 and KO 59). GAPDH was used as a loading control. (H) qRT-PCR analysis of FoxO6 expression in IENS-GFP Foxg1 knock-out clonal lines, compared to parental IENS-GFP (in which log 2 (FC) = 0). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05.

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) qRT-PCR analysis of FOXG1 transgene and endogenous FoxO6 expression in two independent adult mouse NSC lines (‘F6’ and ‘F11-19’) with Dox-inducible FOXG1-V5 expression grown in NSC media with or without Dox for 24 h. Expression shown relative to -Dox (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments, respectively. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05. (B) Schematic of HDR-mediated knock-in of an HA epitope tag at the 3’ end of last FoxO6 coding exon in F6 cells. PCR genotyping of the bulk transfected F6 cell population revealed a 196 bp product, indicating the presence of cells with insertion of the HA tag at the 3’ end of FoxO6. (C) Wide-field immunofluorescent images following immunocytochemistry (ICC) of FoxO6-HA (red) and DAPI (blue) in the tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. Scale bar: 100 μm. (D) Western immunoblot analysis of FOXG1-V5 and FoxO6-HA protein expression in tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. GAPDH was used as a loading control. (E) Experimental strategy for Foxg1 deletion in IENS-GFP cells. Yellow triangles show the target sites of the sgRNAs at either the 5’ or 3’ end of the coding exon. PCR genotyping of parental IENS-GFP cells and Foxg1 KO clonal cell lines (KO 58 and KO 59). Wildtype PCR product ~2.6 kb, knockout PCR product ~1.3 kb. (F) ICC analysis confirms loss of FOXG1 protein expression in IENS-GFP Foxg1 KO clonal lines (KO 58 and KO 59). Scale bar: 100 μm. (G) Western immunoblot confirming loss of Foxg1 protein in the two independent IENS-GFP knock-out clonal lines (KO 58 and KO 59). GAPDH was used as a loading control. (H) qRT-PCR analysis of FoxO6 expression in IENS-GFP Foxg1 knock-out clonal lines, compared to parental IENS-GFP (in which log 2 (FC) = 0). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05.

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Knock-In, Transfection, Immunocytochemistry, Western Blot, Knock-Out

    (A) Schematic of the experimental design for assessing FOXG1-induced reactivation of quiescent NSCs and associated changes in gene expression, using clonal ‘F6’ adult mouse NSC line with Dox-inducible FOXG1-V5 expression. Non-BMP4 treated control = cells in NSC media with EGF/FGF-2. (B) ICC for V5, confirming FOXG1-V5 expression upon Dox addition (Scale bar: 100 μm). (C) Representative phase-contrast images showing changes in cell morphology upon addition of Dox (Scale bar: 100 μm). (D) Colony formation after 24 h BMP4 treatment followed by 10 days in EGF/FGF-2 with or without Dox. Representative images shown of wells stained with methylene blue and imaged on a bright-field microscope. n=3 independent experiments. (E) Higher magnification phase-contrast images of representative colonies after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox as in panel (D) (Scale bar: 200 μm). (F) Number of colonies formed after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox. Mean +/− SD, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (G) qRT-PCR analysis of human FOXG1 transgene and (H) FoxO6 expression during the reactivation time course. Pink = + Dox addition, Blue = No Dox addition. Expression shown relative to non BMP4-treated (EGF/FGF-2) control (in which log 2 (FC)= 0 (dotted line)). Day 0 = expression after 24 h BMP4 treatment. Mean +/− SEM. n = 2 ( FOXG1 ) or 4 ( FoxO6 ). Each data point shows the mean of one experiment performed in technical duplicates. **** P≤0.0001. Two way Anova with Sidak correction.

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) Schematic of the experimental design for assessing FOXG1-induced reactivation of quiescent NSCs and associated changes in gene expression, using clonal ‘F6’ adult mouse NSC line with Dox-inducible FOXG1-V5 expression. Non-BMP4 treated control = cells in NSC media with EGF/FGF-2. (B) ICC for V5, confirming FOXG1-V5 expression upon Dox addition (Scale bar: 100 μm). (C) Representative phase-contrast images showing changes in cell morphology upon addition of Dox (Scale bar: 100 μm). (D) Colony formation after 24 h BMP4 treatment followed by 10 days in EGF/FGF-2 with or without Dox. Representative images shown of wells stained with methylene blue and imaged on a bright-field microscope. n=3 independent experiments. (E) Higher magnification phase-contrast images of representative colonies after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox as in panel (D) (Scale bar: 200 μm). (F) Number of colonies formed after 24 h BMP4 treatment and 10 days in EGF/FGF-2 with or without Dox. Mean +/− SD, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (G) qRT-PCR analysis of human FOXG1 transgene and (H) FoxO6 expression during the reactivation time course. Pink = + Dox addition, Blue = No Dox addition. Expression shown relative to non BMP4-treated (EGF/FGF-2) control (in which log 2 (FC)= 0 (dotted line)). Day 0 = expression after 24 h BMP4 treatment. Mean +/− SEM. n = 2 ( FOXG1 ) or 4 ( FoxO6 ). Each data point shows the mean of one experiment performed in technical duplicates. **** P≤0.0001. Two way Anova with Sidak correction.

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: Expressing, Staining, Microscopy, Quantitative RT-PCR

    (A) Schematic of FoxO6 locus following CRISPR/Cas9-mediated knockout strategy. Yellow triangles show the sgRNA target sites, resulting in a 178 bp deletion in allele 1 in FoxO6 KO clone 53. Exon 1 of allele 2 is replaced by an EF1a-puromycin cassette. (B) PCR genotyping of FoxO6 KO clonal cell lines 6, 53 and 62. PCR 1 and PCR 2, across the 5’ and 3’ homology arms of the EF1a-puromycin cassette, respectively, show correct integration at one of the FoxO6 alleles. PCR 3 shows a 178 bp deletion (53) or loss (6, 62) of the remaining FoxO6 allele. WT parental band = 565 bp, knockout (53) band = 387 bp. (C) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell lines 6 and 62, compared to parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (D) EdU incorporation assay (24h pulse) in parental and FoxO6 KO clonal lines (6 and 62) grown in EGF/FGF-2. (Left) Representative fluorescent images of EdU incorporation. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle marker ( Cdk4, Plk1, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) marker expression in ANS4 parental and FoxO6 KO clonal cell lines (6 and 62) in EGF/FGF-2 and after 24 h BMP4 treatment. Expression values were normalised to Gapdh and shown relative to the expression in parental cells in EGF/FGF-2 (in which log 2 (FC) = 0, shown by the dotted line). Graph shows Mean +/− SD. One experiment, performed in technical duplicates.

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) Schematic of FoxO6 locus following CRISPR/Cas9-mediated knockout strategy. Yellow triangles show the sgRNA target sites, resulting in a 178 bp deletion in allele 1 in FoxO6 KO clone 53. Exon 1 of allele 2 is replaced by an EF1a-puromycin cassette. (B) PCR genotyping of FoxO6 KO clonal cell lines 6, 53 and 62. PCR 1 and PCR 2, across the 5’ and 3’ homology arms of the EF1a-puromycin cassette, respectively, show correct integration at one of the FoxO6 alleles. PCR 3 shows a 178 bp deletion (53) or loss (6, 62) of the remaining FoxO6 allele. WT parental band = 565 bp, knockout (53) band = 387 bp. (C) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell lines 6 and 62, compared to parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (D) EdU incorporation assay (24h pulse) in parental and FoxO6 KO clonal lines (6 and 62) grown in EGF/FGF-2. (Left) Representative fluorescent images of EdU incorporation. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle marker ( Cdk4, Plk1, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) marker expression in ANS4 parental and FoxO6 KO clonal cell lines (6 and 62) in EGF/FGF-2 and after 24 h BMP4 treatment. Expression values were normalised to Gapdh and shown relative to the expression in parental cells in EGF/FGF-2 (in which log 2 (FC) = 0, shown by the dotted line). Graph shows Mean +/− SD. One experiment, performed in technical duplicates.

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: CRISPR, Knock-Out, Quantitative RT-PCR, Expressing, Marker

    (A) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell line 53, compared to ANS4 parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (B) Representative phase-contrast images showing typical NSC morphology in parental and FoxO6 KO 53 cells. Scale bar: 25 μm. (C) Growth curve analysis of parental and FoxO6 KO 53 clonal cells in EGF/FGF-2. Light grey = parental, black = FoxO6 KO 53. Mean +/− SD, n=3 technical replicates. Representative of n=4 independent experiments. Graph showing the gradient of the linear portion of the logistic growth curve (% / h). Mean +/− SEM, n=4 independent experiments. Two-tailed paired Student’s t-test. * P ≤ 0.05. (D) EdU incorporation assay (24h pulse) in EGF/FGF-2 for parental and FoxO6 KO 53 cells. (Left) Representative fluorescent images of EdU incorporation after 24 h pulse. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) Brightfield images of colony formation by parental or FoxO6 KO 53 cells 10 days after plating at low density in NSC media (EGF/FGF-2). Plates stained with methylene blue. (F) Representative phase-contrast images showing morphology of ANS4 and FoxO6 KO 53 after 24h BMP4 treatment. Scale bar: 25 μm. (G) ICC analysis of Nestin and Gfap expression in ANS4 parental and FoxO6 KO 53 cells in EGF/FGF-2 or 3 days BMP4 treatment at low density. Scale bar: 100 μm. (H) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle ( Plk1, Cdk4, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) markers, in parental and FoxO6 KO 53 cells in EGF/FGF-2 and after 24 h BMP4 treatment. Expression shown relative to parental in EGF/FGF-2 (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. (I) EdU incorporation after treatment with BMP4 or EGF/FGF-2 for 24h, followed by a 24h EdU pulse in EGF/FGF-2. Mean +/− SEM. n=2 independent experiments. Each data point shows the mean one experiment, performed in technical triplicates.

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) qRT-PCR analysis of FoxO6 mRNA levels in FoxO6 KO clonal cell line 53, compared to ANS4 parental cells (in which log 2 (FC) = 0). Expression values were normalised to Gapdh. Y axis represents log 2 (Fold change). Mean +/− SEM. n=3 independent experiments. Each data point shows the mean of one experiment, performed in technical duplicates. (B) Representative phase-contrast images showing typical NSC morphology in parental and FoxO6 KO 53 cells. Scale bar: 25 μm. (C) Growth curve analysis of parental and FoxO6 KO 53 clonal cells in EGF/FGF-2. Light grey = parental, black = FoxO6 KO 53. Mean +/− SD, n=3 technical replicates. Representative of n=4 independent experiments. Graph showing the gradient of the linear portion of the logistic growth curve (% / h). Mean +/− SEM, n=4 independent experiments. Two-tailed paired Student’s t-test. * P ≤ 0.05. (D) EdU incorporation assay (24h pulse) in EGF/FGF-2 for parental and FoxO6 KO 53 cells. (Left) Representative fluorescent images of EdU incorporation after 24 h pulse. (Right) Plot shows mean +/− SEM, n=3 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. (E) Brightfield images of colony formation by parental or FoxO6 KO 53 cells 10 days after plating at low density in NSC media (EGF/FGF-2). Plates stained with methylene blue. (F) Representative phase-contrast images showing morphology of ANS4 and FoxO6 KO 53 after 24h BMP4 treatment. Scale bar: 25 μm. (G) ICC analysis of Nestin and Gfap expression in ANS4 parental and FoxO6 KO 53 cells in EGF/FGF-2 or 3 days BMP4 treatment at low density. Scale bar: 100 μm. (H) qRT-PCR analysis of NSC ( Nestin, Olig2, Egfr ), cell cycle ( Plk1, Cdk4, Cmyc ), and astrocyte/quiescence ( Gfap, Aqp4, Id1, Cd9 ) markers, in parental and FoxO6 KO 53 cells in EGF/FGF-2 and after 24 h BMP4 treatment. Expression shown relative to parental in EGF/FGF-2 (in which log 2 (FC) = 0). Mean +/− SEM. n=2/3 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. (I) EdU incorporation after treatment with BMP4 or EGF/FGF-2 for 24h, followed by a 24h EdU pulse in EGF/FGF-2. Mean +/− SEM. n=2 independent experiments. Each data point shows the mean one experiment, performed in technical triplicates.

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Staining

    (A) qRT-PCR analysis of FOXG1 transgene expression in parental or FoxO6 KO 53 cells engineered with inducible FOXG1-V5 after 24 h BMP4 and return to EGF/FGF-2 with our without Dox for 4 days. Expression shown relative parental non-BMP treated (EGF/FGF-2) control (in which log 2 (FC) = 0 (dotted line)). Mean +/− SEM. n=5 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed paired t-test. (B) Representative ICC images showing FOXG1-V5 expression after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox, in parental and FoxO6 KO 53 cells with inducible FOXG1-V5. Scale bar: 100 μm. (C) Percentage of parental or FoxO6 KO 53 cells with inducible FOXG1 expressing FOXG1-V5 (assessed by ICC) after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox. Mean +/− SEM. n = 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Twotailed paired t-test. (D) Representative images of colony formation assay with parental and FoxO6 KO 53 cells at day 10 in EGF/FGF-2, with or without Dox. Plates stained with methylene blue following fixation. (E) Numbers of colonies formed after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM, n= 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Two tailed paired Student’s t-tests. ** P ≤ 0.01. (F) Percentage of the well area covered by cells after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM. n=4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates.

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) qRT-PCR analysis of FOXG1 transgene expression in parental or FoxO6 KO 53 cells engineered with inducible FOXG1-V5 after 24 h BMP4 and return to EGF/FGF-2 with our without Dox for 4 days. Expression shown relative parental non-BMP treated (EGF/FGF-2) control (in which log 2 (FC) = 0 (dotted line)). Mean +/− SEM. n=5 independent experiments. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed paired t-test. (B) Representative ICC images showing FOXG1-V5 expression after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox, in parental and FoxO6 KO 53 cells with inducible FOXG1-V5. Scale bar: 100 μm. (C) Percentage of parental or FoxO6 KO 53 cells with inducible FOXG1 expressing FOXG1-V5 (assessed by ICC) after 24 h BMP4 and 4 days in EGF/FGF-2, with or without Dox. Mean +/− SEM. n = 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Twotailed paired t-test. (D) Representative images of colony formation assay with parental and FoxO6 KO 53 cells at day 10 in EGF/FGF-2, with or without Dox. Plates stained with methylene blue following fixation. (E) Numbers of colonies formed after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM, n= 4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates. Two tailed paired Student’s t-tests. ** P ≤ 0.01. (F) Percentage of the well area covered by cells after 24 h BMP4 and 10-15 days in EGF/FGF-2, with or without Dox as in panel (D). Mean +/− SEM. n=4 independent experiments. Each data point shows the mean of one experiment performed in technical triplicates.

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Colony Assay, Staining

    (A) Phase contrast imaging following Dox addition to untransfected ANS4 cells does not induce vacuole formation. Scale bar 100 um. (B) ICC following Dox-induced (24h) MYEF2-HA-IRES-MCHERRY overexpression in mouse GSC line ‘NPE’ shows no evidence of vacuole formation. Scale bar 50 um. (C) BODIPY lipid staining does not colocalise with vacuole structures in FoxO6-inducible cell line (C71, 2 days +/− Dox). Scale bar 25um. (D) EGF-647 uptake after a pulse of 1 hr shows puncta representative of receptor-mediated endocytosis (C71 incubated overnight with Dox prior to EGF-647 pulse). Scale bar 25 um. (E) Western blot analysis of LAMP1, EEA1 and HA upon FoxO6-HA overexpression (+/− Dox). GAPDH is used as a loading control. Bulk transfected population sorted for mCherry and clonal cell lines (6, 41, 59, 71) analysed. (F) Phase contrast images show vacuole formation in FoxO6-HA inducible cell lines following Dox+Dextran overnight incubation, prior to flow cytometry analysis. Scale bar 100 um. (G) Imaging of EdU incorporation in FoxO6-HA inducible cells (C71) after 2 days in EGF/FGF +/− Dox (24h pulse). Scale bar 100 um or 25um. (H) EdU incorporation after EGF/FGF-2 or BMP4 for 3 days +/− Dox (24h pulse). n=3 technical replicates, mean +/− SD. (I) Phase-contrast images of Dox treated cells in culture (c71). Vacuolated cells remain after 5 days in Dox and following Dox removal. (J) ICC in F6 cells with Dox-inducible FOXG1-V5 shows no evidence of vacuolisation upon Dox addition (FOXG1-V5 induction). Scale bar 100 um.

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) Phase contrast imaging following Dox addition to untransfected ANS4 cells does not induce vacuole formation. Scale bar 100 um. (B) ICC following Dox-induced (24h) MYEF2-HA-IRES-MCHERRY overexpression in mouse GSC line ‘NPE’ shows no evidence of vacuole formation. Scale bar 50 um. (C) BODIPY lipid staining does not colocalise with vacuole structures in FoxO6-inducible cell line (C71, 2 days +/− Dox). Scale bar 25um. (D) EGF-647 uptake after a pulse of 1 hr shows puncta representative of receptor-mediated endocytosis (C71 incubated overnight with Dox prior to EGF-647 pulse). Scale bar 25 um. (E) Western blot analysis of LAMP1, EEA1 and HA upon FoxO6-HA overexpression (+/− Dox). GAPDH is used as a loading control. Bulk transfected population sorted for mCherry and clonal cell lines (6, 41, 59, 71) analysed. (F) Phase contrast images show vacuole formation in FoxO6-HA inducible cell lines following Dox+Dextran overnight incubation, prior to flow cytometry analysis. Scale bar 100 um. (G) Imaging of EdU incorporation in FoxO6-HA inducible cells (C71) after 2 days in EGF/FGF +/− Dox (24h pulse). Scale bar 100 um or 25um. (H) EdU incorporation after EGF/FGF-2 or BMP4 for 3 days +/− Dox (24h pulse). n=3 technical replicates, mean +/− SD. (I) Phase-contrast images of Dox treated cells in culture (c71). Vacuolated cells remain after 5 days in Dox and following Dox removal. (J) ICC in F6 cells with Dox-inducible FOXG1-V5 shows no evidence of vacuolisation upon Dox addition (FOXG1-V5 induction). Scale bar 100 um.

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: Imaging, Over Expression, Staining, Incubation, Western Blot, Transfection, Flow Cytometry

    (A) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells with Dox-inducible FOXG1-V5 grown in EGF/FGF-2 for 24h plus or minus Dox (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates). (B) Western blot analysis of Pak1 expression in F6 cells treated with Dox in EGF/FGF for 24h. GAPDH is used as a loading control. Quantification of Pak1 bands normalised to GAPDH and -Dox control shown, where -Dox =1. (C) qRT-PCR analysis of Pak1 expression in ANS4 parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates.) Two-tailed one sample t-test, * p<0.05. (D) Western blot analysis of Pak1 expression in parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates). (E) Quantification of Pak1 Western blot band intensities as in panel (D). Parental = 1 as shown by the dotted line (n=3 biological replicates, Mean +/− SEM. Each data point shows the intensity from one experiment). Two-tailed one sample t-test, * p<0.05. (F) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells after 24 h BMP4 and return to EGF/FGF-2 with or without Dox for 2 days. Expression shown relative non BMP-treated (EGF/FGF-2) control (in which log 2 (FC) = 0). Day 0 = expression after 24 h BMP4 treatment (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates).

    Journal: bioRxiv

    Article Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

    doi: 10.1101/2022.06.30.498283

    Figure Lengend Snippet: (A) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells with Dox-inducible FOXG1-V5 grown in EGF/FGF-2 for 24h plus or minus Dox (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates). (B) Western blot analysis of Pak1 expression in F6 cells treated with Dox in EGF/FGF for 24h. GAPDH is used as a loading control. Quantification of Pak1 bands normalised to GAPDH and -Dox control shown, where -Dox =1. (C) qRT-PCR analysis of Pak1 expression in ANS4 parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates.) Two-tailed one sample t-test, * p<0.05. (D) Western blot analysis of Pak1 expression in parental versus FoxO6 KO clones 6, 53 and 62 (n=3 biological replicates). (E) Quantification of Pak1 Western blot band intensities as in panel (D). Parental = 1 as shown by the dotted line (n=3 biological replicates, Mean +/− SEM. Each data point shows the intensity from one experiment). Two-tailed one sample t-test, * p<0.05. (F) qRT-PCR analysis of FOXG1 transgene, and endogenous FoxO6 and Pak1 expression in F6 cells after 24 h BMP4 and return to EGF/FGF-2 with or without Dox for 2 days. Expression shown relative non BMP-treated (EGF/FGF-2) control (in which log 2 (FC) = 0). Day 0 = expression after 24 h BMP4 treatment (n=2 biological replicates, Mean +/− SEM. Each data point shows the mean of one experiment performed in technical duplicates).

    Article Snippet: This media consists of DMEM/HAMS-F12 (Sigma D8437) supplemented with N2 and B27 (Life Technologies/Gibco), penicillin, streptomycin (Gibco), BSA (Gibco), b-mercaptoethanol (Gibco), MEM NEAA (Gibco), 1 μg/ml Laminin (Sigma or Cultrex), 10 ng/ml mouse EGF and 10 ng/ml human FGF-2 (Peprotech) Media was exchanged every 3-4 days.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Clone Assay, Two Tailed Test

    Factors screened by tubule culture experiments for their roles in the proliferation and differentiation of stem Leydig cells

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes

    doi: 10.1073/pnas.1519395113

    Figure Lengend Snippet: Factors screened by tubule culture experiments for their roles in the proliferation and differentiation of stem Leydig cells

    Article Snippet: This same sequence occurs in vivo ( 21 , 22 ). table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Reagent Signaling pathway Concentration (range tested) Manufacturer Ovine LH 10 ng/mL (0.1–10) Hormone Program USDA Rat FSH 20 ng/mL (0.2–20) Hormone Program NIDDK T3 10 nM (0.1–10) Sigma-Aldrich Estradiol (E2) 10 ng/mL (0.1–10) Steraloids DHT 10 ng/mL (0.1–10) Steraloids R1881 AR − 10 nM (0.1–10) Sigma-Aldrich Dexamethasone 10 nM (0.1–10) Sigma-Aldrich Retinoic acid 1 μM (0.01–1) Sigma-Aldrich Mouse PDGFAA 10 ng/mL (0.1–10) Prospec Protein Specialists Rat PDGFBB 10 ng/mL (0.1–10) Sigma-Aldrich PDGFR Inhibitor IV PDGFBB − 20 nM (0.2–20) EMD Bioscience Imatinib Mesylate c-Kit − /PDGFR − 0.2 μM (0.02–2) Selleck Chemicals Human TGFβ1 10 ng/mL (0.1–10) Cell Signaling Technology Human Activin 10 ng/mL (0.1–10) Sigma-Aldrich SB431542 TGFβ − /Activin − 0.1 μM (0.01–1) Selleck Chemicals SB525334 TGF-β − 50 nM (0.5–50) Selleck Chemicals Rat TGFα 10 ng/mL (0.1–10) Sigma-Aldrich Mouse EGF 10 ng/mL (0.1–10) Sigma-Aldrich Porcine Inhibin 10 ng/mL (0.1–10) Sigma-Aldrich Rat IL-1β 1 ng/mL (0.1–10) Sigma-Aldrich Rat IGF1 50 ng/mL (0.5–50) Sigma-Aldrich Human SCF 10 ng/mL (0.1–10) MP Biomedicals Rat GDNF 10 ng/mL (0.1–10) EMD Chemicals Human FGF2 10 ng/mL (0.1–10) Novus Biologiclas Mouse LIF 10 ng/mL (0.1–10) Sigma-Aldrich DAPT Notch − 2 μM (0.2–20) EMD Bioscience Lithium ?

    Techniques: